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Ectomycorrhizal plants trade plant-assimilated carbon for soil nutrients with their fungal partners. The underlying mechanisms, however, are not fully understood. Here we investigate the exchange of carbon for nitrogen in the ectomycorrhizal symbiosis of Fagus sylvatica across different spatial scales from the root system to the cellular level. We provided 15N-labelled nitrogen to mycorrhizal hyphae associated with one half of the root system of young beech trees, while exposing plants to a 13CO2 atmosphere. We analysed the short-term distribution of 13C and 15N in the root system with isotope-ratio mass spectrometry, and at the cellular scale within a mycorrhizal root tip with nanoscale secondary ion mass spectrometry (NanoSIMS). At the root system scale, plants did not allocate more 13C to root parts that received more 15N. Nanoscale secondary ion mass spectrometry imaging, however, revealed a highly heterogenous, and spatially significantly correlated distribution of 13C and 15N at the cellular scale. Our results indicate that, on a coarse scale, plants do not allocate a larger proportion of photoassimilated C to root parts associated with N-delivering ectomycorrhizal fungi. Within the ectomycorrhizal tissue, however, recently plant-assimilated C and fungus-delivered N were spatially strongly coupled. Here, NanoSIMS visualisation provides an initial insight into the regulation of ectomycorrhizal C and N exchange at the microscale.

Research needs a balance of risk-taking in "breakthrough projects" and gradual progress. For building a sustainable knowledge base, it is indispensable to provide support for both.

Grassland ecosystems cover around 37% of the ice-free land surface on Earth and have critical socioeconomic importance globally. As in many terrestrial ecosystems, biological dinitrogen (N2) fixation represents an essential natural source of nitrogen (N). The ability to fix atmospheric N2 is limited to diazotrophs, a diverse guild of bacteria and archaea. To elucidate the abiotic (climatic, edaphic), biotic (vegetation), and spatial factors that govern diazotrophic community composition in global grassland soils, amplicon sequencing of the dinitrogenase reductase gene—nifH—was performed on samples from a replicated standardized nutrient [N, phosphorus (P)] addition experiment in 23 grassland sites spanning four continents. Sites harbored distinct and diverse diazotrophic communities, with most of reads assigned to diazotrophic taxa within the Alphaproteobacteria (e.g., Rhizobiales), Cyanobacteria (e.g., Nostocales), and Deltaproteobacteria (e.g., Desulforomonadales) groups. Likely because of the wide range of climatic and edaphic conditions and spatial distance among sampling sites, only a few of the taxa were present at all sites. The best model describing the variation among soil diazotrophic communities at the OTU level combined climate seasonality (temperature in the wettest quarter and precipitation in the warmest quarter) with edaphic (C:N ratio, soil texture) and vegetation factors (various perennial plant covers). Additionally, spatial variables (geographic distance) correlated with diazotrophic community variation, suggesting an interplay of environmental variables and spatial distance. The diazotrophic communities appeared to be resilient to elevated nutrient levels, as 2–4 years of chronic N and P additions had little effect on the community composition. However, it remains to be seen, whether changes in the community composition occur after exposure to long-term, chronic fertilization regimes.

High-affinity terminal oxidases (TOs) are believed to permit microbial respiration at low oxygen (O-2) levels. Genes encoding such oxidases are widespread, and their existence in microbial genomes is taken as an indicator for microaerobic respiration. We combined respiratory kinetics determined via highly sensitive optical trace O-2 sensors, genomics, and transcriptomics to test the hypothesis that high-affinity TOs are a prerequisite to respire micro- and nanooxic concentrations of O-2 in environmentally relevant model soil organisms: acidobacteria. Members of the Acidobacteria harbor branched respiratory chains terminating in low-affinity (caa(3)-type cytochrome c oxidases) as well as high-affinity (cbb(3)-type cytochrome c oxidases and/or bd-type quinol oxidases) TOs, potentially enabling them to cope with varying O(2 )concentrations. The measured apparent K-m (K-m(app())) values for O(2 )of selected strains ranged from 37 to 288 nmol O(2 )liter(-1), comparable to values previously assigned to low-affinity TOs. Surprisingly, we could not detect the expression of the conventional high-affinity TO (cbb3 type) at micro- and nanomolar O(2 )concentrations but detected the expression of low-affinity TOs. To the best of our knowledge, this is the first observation of microaerobic respiration imparted by low-affinity TOs at O-2 concentrations as low as 1 nM. This challenges the standing hypothesis that a microaerobic lifestyle is exclusively imparted by the presence of high-affinity TOs. As low-affinity TOs are more efficient at generating ATP than high-affinity TOs, their utilization could provide a great benefit, even at low-nanomolar O(2 )levels. Our findings highlight energy conservation strategies that could promote the success of Acidobacteria in soil but might also be important for as-yet-unrevealed microorganisms., mSystems, 6 (4), ISSN:2379-5077

34 Pags.- 9 Figs. The definitive version is available at: https://onlinelibrary.wiley.com/journal/13653040, Legume–rhizobia symbioses play a major role in food production for an ever growing human population. In this symbiosis, dinitrogen is reduced (“fixed”) to ammonia by the rhizobial nitrogenase enzyme complex and is secreted to the plant host cells, whereas dicarboxylic acids derived from photosynthetically produced sucrose are transported into the symbiosomes and serve as respiratory substrates for the bacteroids. The symbiosome membrane contains high levels of SST1 protein, a sulfate transporter. Sulfate is an essential nutrient for all living organisms, but its importance for symbiotic nitrogen fixation and nodule metabolism has long been underestimated. Using chemical imaging, we demonstrate that the bacteroids take up 20‐fold more sulfate than the nodule host cells. Furthermore, we show that nitrogenase biosynthesis relies on high levels of imported sulfate, making sulfur as essential as carbon for the regulation and functioning of symbiotic nitrogen fixation. Our findings thus establish the importance of sulfate and its active transport for the plant–microbe interaction that is most relevant for agriculture and soil fertility., This work was funded by the Austrian Science Fund (DK plus, W 1257‐820) and COST action FA1306 to S.W. and by grant AGL2017‐85775‐R from Ministerio de Economía y Competitividad‐Fondos Europeos de Desarrollo Regional (Spain) to M.B.

Hypersaline microbial mats are dense microbial ecosystems capable of performing complete element cycling and are considered analogs of early Earth and hypothetical extraterrestrial ecosystems. We studied the functionality and limits of key biogeochemical processes, such as photosynthesis, aerobic respiration, and sulfur cycling, in salt crust-covered microbial mats from a tidal flat at the coast of Oman. We measured light, oxygen, and sulfide microprofiles as well as sulfate reduction rates at salt saturation and in flood conditions and determined fine-scale stratification of pigments, biomass, and microbial taxa in the resident microbial community. The salt crust did not protect the mats against irradiation or evaporation. Although some oxygen production was measurable at salinities of ≤30% (wt/vol) in situ, at saturation-level salinity (40%), oxygenic photosynthesis was completely inhibited and only resumed 2 days after reducing the porewater salinity to 12%. Aerobic respiration and active sulfur cycling occurred at low rates under salt saturation and increased strongly upon salinity reduction. Apart from high relative abundances of Chloroflexi, photoheterotrophic Alphaproteobacteria, Bacteroidetes, and Archaea, the mat contained a distinct layer harboring filamentous Cyanobacteria, which is unusual for such high salinities. Our results show that the diverse microbial community inhabiting this salt flat mat ultimately depends on periodic salt dilution to be self-sustaining and is rather adapted to merely survive salt saturation than to thrive under the salt crust. IMPORTANCE Due to their abilities to survive intense radiation and low water availability, hypersaline microbial mats are often suggested to be analogs of potential extraterrestrial life. However, even the limitations imposed on microbial processes by saturation-level salinity found on Earth have rarely been studied in situ. While abundance and diversity of microbial life in salt-saturated environments are well documented, most of our knowledge on process limitations stems from culture-based studies, few in situ studies, and theoretical calculations. In particular, oxygenic photosynthesis has barely been explored beyond 5 M NaCl (28% wt/vol). By applying a variety of biogeochemical and molecular methods, we show that despite abundance of photoautotrophic microorganisms, oxygenic photosynthesis is inhibited in salt-crust-covered microbial mats at saturation salinities, while rates of other energy generation processes are decreased several-fold. Hence, the complete element cycling required for self-sustaining microbial communities only occurs at lower salt concentrations, Applied and Environmental Microbiology, 87 (17), ISSN:0099-2240, ISSN:1098-5336

Significant rates of atmospheric dihydrogen (H2) consumption have been observed in temperate soils due to the activity of high-affinity enzymes, such as the group 1h [NiFe]-hydrogenase. We designed broadly inclusive primers targeting the large subunit gene (hhyL) of group 1h [NiFe]-hydrogenases for long-read sequencing to explore its taxonomic distribution across soils. This approach revealed a diverse collection of microorganisms harboring hhyL, including previously unknown groups and taxonomically not assignable sequences. Acidobacterial group 1h [NiFe]-hydrogenase genes were abundant and expressed in temperate soils. To support the participation of acidobacteria in H2 consumption, we studied two representative mesophilic soil acidobacteria, which expressed group 1h [NiFe]-hydrogenases and consumed atmospheric H2 during carbon starvation. This is the first time mesophilic acidobacteria, which are abundant in ubiquitous temperate soils, have been shown to oxidize H2 down to below atmospheric concentrations. As this physiology allows bacteria to survive periods of carbon starvation, it could explain the success of soil acidobacteria. With our long-read sequencing approach of group 1h [NiFe]-hydrogenase genes, we show that the ability to oxidize atmospheric levels of H2 is more widely distributed among soil bacteria than previously recognized and could represent a common mechanism enabling bacteria to persist during periods of carbon deprivation.

Significant rates of atmospheric dihydrogen (H2) consumption have been observed in temperate soils due to the activity of high-affinity enzymes, such as the group 1h [NiFe]-hydrogenase. We designed broadly inclusive primers targeting the large subunit gene (hhyL) of group 1h [NiFe]-hydrogenases for long-read sequencing to explore its taxonomic distribution across soils. This approach revealed a diverse collection of microorganisms harboring hhyL, including previously unknown groups and taxonomically not assignable sequences. Acidobacterial group 1h [NiFe]-hydrogenase genes were abundant and expressed in temperate soils. To support the participation of acidobacteria in H2 consumption, we studied two representative mesophilic soil acidobacteria, which expressed group 1h [NiFe]-hydrogenases and consumed atmospheric H2 during carbon starvation. This is the first time mesophilic acidobacteria, which are abundant in ubiquitous temperate soils, have been shown to oxidize H2 down to below atmospheric concentrations. As this physiology allows bacteria to survive periods of carbon starvation, it could explain the success of soil acidobacteria. With our long-read sequencing approach of group 1h [NiFe]-hydrogenase genes, we show that the ability to oxidize atmospheric levels of H2 is more widely distributed among soil bacteria than previously recognized and could represent a common mechanism enabling bacteria to persist during periods of carbon deprivation.

Our current understanding of microbial hotspots such as the rhizosphere mainly stems from observations through measurements at the macroscopic scale, integrating a multitude of microbial cells and taxa into a few measured variables. Consequently, we still lack an understanding of the individual participants that actively contribute to processes. Identifying microorganisms and relating their activity to these processes within the soil-plant interface on a microscopic scale represent a missing link in understanding nutrient flux in agriculturally important ecosystems such as rice cultivation.I will present a novel workflow for single-cell isotope imaging in the rhizosphere that combines fluorescence in situ hybridization, gold-targeted secondary electron microscopy, and nano-scale secondary ion mass spectrometry. Based on correlative microscopy and hotspot detection, this approach now allows to (i) identify single bacteria on root surfaces that actively incorporate stable isotopes, (ii) quantify their contribution to processes of interest within a given population, and (iii) potentially trace nutrient fluxes among plants and bacteria on a microscale.Illuminating plant-microorganism interactions on a microscale provides the potential to evaluate the actual impact of bio-inoculants applied as fertilizers and to engineer plant-microorganism associations which may be essential to increase the production of major staple crops for a growing world population.

As the global hydrological cycle intensifies with future warming, more severe droughts will alter the terrestrial biogeochemical carbon (C) cycle. As soil microbial physiology controls the large fluxes of C from soil to the atmosphere, improving our ability to accurately quantify microbial physiological parameters in soil is essential. However, currently available methods to determine microbial C metabolism in soil require the addition of water, which makes it practically impossible to measure microbial physiology in dry soil samples without stimulating microbial growth and respiration (namely, the “Birch effect”).We developed a new method based on in vivo 18O water vapor equilibration to minimize soil re-wetting effects. This method allows the isotopic labelling of soil water without any liquid water or dissolved substrate addition to the sample. This was compared to the main current method (18O-water application method) in soil samples either at near-optimal water holding capacity or in air dry soils. We generated time curves of the isotopic equilibration between liquid soil water and water vapor and calculated the average atom percent 18O excess over incubation time, which is necessary to calculate microbial growth rates. We tested isotopic equilibration patterns in nine different soils (natural and artificially constructed ones) covering a wide range of soil texture and organic matter content. We then measured microbial growth, respiration and carbon use efficiency in three natural soils (either dry or at near-optimal water holding capacity). The proposed 18O vapor equilibration method provides similar results as the currently widely used method of liquid 18O water addition to determine microbial growth when used a near-optimal water holding capacity. However, when applied to dry soils the liquid 18O water addition method overestimated growth by up to 250%, respiration by up to 500%, and underestimated carbon use efficiency by up to 40%.Finally, we applied the new method to undisturbed biocrust samples, at field water content (1-3%), and show for the first time real microbial growth rates and CUE values in such arid ecosystems. We describe new insights into biogeochemical cycling of C that the new method can help uncover and consider the wide range of questions regarding microbial physiology and its response to global change that can now be proposed and addressed.

Nitrification is a ubiquitous microbially mediated process in the environment and an essential process in engineered systems such as wastewater and drinking water treatment plants. However, nitrification also contributes to fertilizer loss from agricultural environments, increasing the eutrophication of downstream aquatic ecosystems, and produces the greenhouse gas nitrous oxide. As ammonia-oxidizing bacteria are the most dominant ammonia-oxidizing microbes in fertilized agricultural soils, understanding their responses to a variety of environmental conditions is essential for curbing the negative environmental effects of nitrification. Notably, oxygen limitation has been reported to significantly increase nitric oxide and nitrous oxide production during nitrification. Here, we investigate the physiology of the best-characterized ammonia-oxidizing bacterium, Nitrosomonas europaea, growing under oxygen-limited conditions., Ammonia-oxidizing microorganisms perform the first step of nitrification, the oxidation of ammonia to nitrite. The bacterium Nitrosomonas europaea is the best-characterized ammonia oxidizer to date. Exposure to hypoxic conditions has a profound effect on the physiology of N. europaea, e.g., by inducing nitrifier denitrification, resulting in increased nitric and nitrous oxide production. This metabolic shift is of major significance in agricultural soils, as it contributes to fertilizer loss and global climate change. Previous studies investigating the effect of oxygen limitation on N. europaea have focused on the transcriptional regulation of genes involved in nitrification and nitrifier denitrification. Here, we combine steady-state cultivation with whole-genome transcriptomics to investigate the overall effect of oxygen limitation on N. europaea. Under oxygen-limited conditions, growth yield was reduced and ammonia-to-nitrite conversion was not stoichiometric, suggesting the production of nitrogenous gases. However, the transcription of the principal nitric oxide reductase (cNOR) did not change significantly during oxygen-limited growth, while the transcription of the nitrite reductase-encoding gene (nirK) was significantly lower. In contrast, both heme-copper-containing cytochrome c oxidases encoded by N. europaea were upregulated during oxygen-limited growth. Particularly striking was the significant increase in transcription of the B-type heme-copper oxidase, proposed to function as a nitric oxide reductase (sNOR) in ammonia-oxidizing bacteria. In the context of previous physiological studies, as well as the evolutionary placement of N. europaea’s sNOR with regard to other heme-copper oxidases, these results suggest sNOR may function as a high-affinity terminal oxidase in N. europaea and other ammonia-oxidizing bacteria. IMPORTANCE Nitrification is a ubiquitous microbially mediated process in the environment and an essential process in engineered systems such as wastewater and drinking water treatment plants. However, nitrification also contributes to fertilizer loss from agricultural environments, increasing the eutrophication of downstream aquatic ecosystems, and produces the greenhouse gas nitrous oxide. As ammonia-oxidizing bacteria are the most dominant ammonia-oxidizing microbes in fertilized agricultural soils, understanding their responses to a variety of environmental conditions is essential for curbing the negative environmental effects of nitrification. Notably, oxygen limitation has been reported to significantly increase nitric oxide and nitrous oxide production during nitrification. Here, we investigate the physiology of the best-characterized ammonia-oxidizing bacterium, Nitrosomonas europaea, growing under oxygen-limited conditions.

We report eight genomes from representatives of the phylum Acidobacteria subdivisions 1 and 3, isolated from soils. The genome sizes range from 4.9 to 6.7 Mb. Genomic analysis reveals putative genes for low- and high-affinity respiratory oxygen reductases, high-affinity hydrogenases, and the capacity to use a diverse collection of carbohydrates.

Microbial life is surprisingly abundant and diverse in global desert ecosystems. In these environments, microorganisms endure a multitude of physicochemical stresses, including low water potential, carbon and nitrogen starvation, and extreme temperatures. In this review, we summarize our current understanding of the energetic mechanisms and trophic dynamics that underpin microbial function in desert ecosystems. Accumulating evidence suggests that dormancy is a common strategy that facilitates microbial survival in response to water and carbon limitation., Microbial life is surprisingly abundant and diverse in global desert ecosystems. In these environments, microorganisms endure a multitude of physicochemical stresses, including low water potential, carbon and nitrogen starvation, and extreme temperatures. In this review, we summarize our current understanding of the energetic mechanisms and trophic dynamics that underpin microbial function in desert ecosystems. Accumulating evidence suggests that dormancy is a common strategy that facilitates microbial survival in response to water and carbon limitation. Whereas photoautotrophs are restricted to specific niches in extreme deserts, metabolically versatile heterotrophs persist even in the hyper-arid topsoils of the Atacama Desert and Antarctica. At least three distinct strategies appear to allow such microorganisms to conserve energy in these oligotrophic environments: degradation of organic energy reserves, rhodopsin- and bacteriochlorophyll-dependent light harvesting, and oxidation of the atmospheric trace gases hydrogen and carbon monoxide. In turn, these principles are relevant for understanding the composition, functionality, and resilience of desert ecosystems, as well as predicting responses to the growing problem of desertification.

Soil microbial carbon use efficiency (CUE), defined as the ratio of organic C allocated to growth over organic C taken up, strongly affects soil carbon (C) cycling. Despite the importance of the microbial CUE for the terrestrial C cycle, very little is known about how it is affected by nutrient availability. Therefore, we studied microbial CUE and microbial biomass turnover time in soils of a long-term fertilization experiment in a temperate grassland comprising five treatments (control, PK, NK, NP, NPK). Microbial CUE and the turnover of microbial biomass were determined using a novel substrate-independent method based on incorporation of 18 O from labeled water into microbial DNA. Microbial respiration was 28–37% smaller in all three N treatments (NK, NP, and NPK) compared to the control, whereas the PK treatment did not affect microbial respiration. N-fertilization decreased microbial C uptake, while the microbial growth rate was not affected. Microbial CUE ranged between 0.31 and 0.45, and was 1.3- to 1.4-fold higher in the N-fertilized soils than in the control. The turnover time ranged between 80 and 113 days and was not significantly affected by fertilization. Net primary production (NPP) and the abundance of legumes differed strongly across the treatments, and the fungal:bacterial ratio was very low in all treatments. Structural equation modeling revealed that microbial CUE was exclusively controlled by N fertilization and that neither the abundance of legumes (as a proxy for the quality of the organic matter inputs) nor NPP (as a proxy for C inputs) had an effect on microbial CUE. Our results show that N fertilization did not only decrease microbial respiration, but also microbial C uptake, indicating that less C was intracellularly processed in the N fertilized soils. The reason for reduced C uptake and increased CUE in the N-fertilization treatments is likely an inhibition of oxidative enzymes involved in the degradation of aromatic compounds by N in combination with a reduced energy requirement for microbial N acquisition in the fertilized soils. In conclusion, the study shows that N availability can control soil C cycling by affecting microbial CUE, while plant community-mediated changes in organic matter inputs and P and K availability played no important role for C partitioning of the microbial community in this temperate grassland.

Soil microorganisms control carbon losses from soils to the atmosphere 13 , yet their responses to climate warming are often short-lived and unpredictable 47 . Two mechanisms, microbial acclimation and substrate depletion, have been proposed to explain temporary warming effects on soil micro- bial activity 810 . However, empirical support for either mecha- nism is unconvincing. Here we used geothermal temperature gradients ( > 50 years of field warming) 11 and a short-term experiment to show that microbial activity (gross rates of growth, turnover, respiration and carbon uptake) is intrinsi- cally temperature sensitive and does not acclimate to warm- ing ( + 6 °C) over weeks or decades. Permanently accelerated microbial activity caused carbon loss from soil. However, soil carbon loss was temporary because substrate depletion reduced microbial biomass and constrained the influence of microbes over the ecosystem. A microbial biogeochemical model 12 14 showed that these observations are reproducible through a modest, but permanent, acceleration in microbial physiology. These findings reveal a mechanism by which intrinsic microbial temperature sensitivity and substrate depletion together dictate warming effects on soil carbon loss via their control over microbial biomass. We thus provide a framework for interpreting the links between temperature, microbial activity and soil carbon loss on timescales relevant to Earths climate system.

Stable isotope labeling of agricultural polyesters enables demonstration of their microbial utilization in soils., Plastic materials are widely used in agricultural applications to achieve food security for the growing world population. The use of biodegradable instead of nonbiodegradable polymers in single-use agricultural applications, including plastic mulching, promises to reduce plastic accumulation in the environment. We present a novel approach that allows tracking of carbon from biodegradable polymers into CO2 and microbial biomass. The approach is based on 13C-labeled polymers and on isotope-specific analytical methods, including nanoscale secondary ion mass spectrometry (NanoSIMS). Our results unequivocally demonstrate the biodegradability of poly(butylene adipate-co-terephthalate) (PBAT), an important polyester used in agriculture, in soil. Carbon from each monomer unit of PBAT was used by soil microorganisms, including filamentous fungi, to gain energy and to form biomass. This work advances both our conceptual understanding of polymer biodegradation and the methodological capabilities to assess this process in natural and engineered environments.

Sulfur-cycling microorganisms impact organic matter decomposition in wetlands and consequently greenhouse gas emissions from these globally relevant environments. However,their identities and physiological properties are largely unknown. By applying a functional metagenomics approach to an acidic peatland, we recovered draft genomes of seven novelAcidobacteriaspecies with the potential for dissimilatory sulfite (dsrAB,dsrC,dsrD,dsrN, dsrT, dsrMKJOP) or sulfate respiration (sat, aprBA, qmoABCplusdsrgenes). Surprisingly, the genomes also encodeddsrL, a unique gene of the sulfur oxidation pathway. Metatranscriptome analysis demonstrated expression of acidobacterial sulfur-metabolism genes in native peat soil and their upregulation in diverse anoxic microcosms. This indicated an active sulfate respiration pathway, which, however, could also operate in reverse for sulfur oxidation as recently shown for other microorganisms.Acidobacteriathat only harbored genes for sulfite reduction additionally encoded enzymes that liberate sulfite from organosulfonates, which suggested organic sulfur compounds as complementary energy sources. Further metabolic potentials included polysaccharide hydrolysis and sugar utilization, aerobic respiration, several fermentative capabilities, and hydrogen oxidation. Our findings extend both, the known physiological and genetic properties ofAcidobacteriaand the known taxonomic diversity of microorganisms with a DsrAB-based sulfur metabolism, and highlight new fundamental niches for facultative anaerobicAcidobacteriain wetlands based on exploitation of inorganic and organic sulfur molecules for energy conservation.

© 2018 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd. Members of the phylum Acidobacteria are abundant and ubiquitous across soils. We performed a large-scale comparative genome analysis spanning subdivisions 1, 3, 4, 6, 8 and 23 (n = 24) with the goal to identify features to help explain their prevalence in soils and understand their ecophysiology. Our analysis revealed that bacteriophage integration events along with transposable and mobile elements influenced the structure and plasticity of these genomes. Low- and high-affinity respiratory oxygen reductases were detected in multiple genomes, suggesting the capacity for growing across different oxygen gradients. Among many genomes, the capacity to use a diverse collection of carbohydrates, as well as inorganic and organic nitrogen sources (such as via extracellular peptidases), was detected – both advantageous traits in environments with fluctuating nutrient environments. We also identified multiple soil acidobacteria with the potential to scavenge atmospheric concentrations of H2, now encompassing mesophilic soil strains within the subdivision 1 and 3, in addition to a previously identified thermophilic strain in subdivision 4. This large-scale acidobacteria genome analysis reveal traits that provide genomic, physiological and metabolic versatility, presumably allowing flexibility and versatility in the challenging and fluctuating soil environment.

To study how microbes establish themselves in a mammalian gut environment, we colonized germ-free mice with microbial communities from human, zebrafish and termite guts, human skin and tongue, soil, and estuarine microbial mats. Bacteria from these foreign environments colonized and persisted in the mouse gut; their capacity to metabolize dietary and host carbohydrates, and bile acids, correlated with colonization success. Co-housing mice harboring these xenomicrobiota with one another, with mice harboring native gut microbiota, and germ-free ‘bystanders’ revealed the success of particular bacterial taxa in colonizing an empty gut habitat and guts with established communities. Unanticipated patterns of ecological succession were observed; for example, a soil-derived bacterium dominated even in the presence of bacteria from other gut communities (zebrafish and termite), and human-derived bacteria colonized germ-free mice before mouse-derived organisms. This approach generalizes to address a variety of mechanistic questions about succession, including succession in the context of microbiota-directed therapeutics.

Contains fulltext : 117292.pdf (Publisher’s version ) (Open Access) Anaerobic ammonium-oxidizing (anammox) bacteria are responsible for a significant portion of the loss of fixed nitrogen from the oceans, making them important players in the global nitrogen cycle. To date, marine anammox bacteria found in marine water columns and sediments worldwide belong almost exclusively to the 'Candidatus Scalindua' species, but the molecular basis of their metabolism and competitive fitness is presently unknown. We applied community sequencing of a marine anammox enrichment culture dominated by 'Candidatus Scalindua profunda' to construct a genome assembly, which was subsequently used to analyse the most abundant gene transcripts and proteins. In the S. profunda assembly, 4756 genes were annotated, and only about half of them showed the highest identity to the only other anammox bacterium of which a metagenome assembly had been constructed so far, the freshwater 'Candidatus Kuenenia stuttgartiensis'. In total, 2016 genes of S. profunda could not be matched to the K. stuttgartiensis metagenome assembly at all, and a similar number of genes in K.stuttgartiensis could not be found in S. profunda. Most of these genes did not have a known function but 98 expressed genes could be attributed to oligopeptide transport, amino acid metabolism, use of organic acids and electron transport. On the basis of the S. profunda metagenome, and environmental metagenome data, we observed pronounced differences in the gene organization and expression of important anammox enzymes, such as hydrazine synthase (HzsAB), nitrite reductase (NirS) and inorganic nitrogen transport proteins. Adaptations of Scalindua to the substrate limitation of the ocean may include highly expressed ammonium, nitrite and oligopeptide transport systems and pathways for the transport, oxidation, and assimilation of small organic compounds that may allow a more versatile lifestyle contributing to the competitive fitness of Scalindua in the marine realm.

The nonheterocystous filamentous cyanobacterium, strain ESFC-1, is a recently described member of the order Oscillatoriales within the Cyanobacteria. ESFC-1 has been shown to be a major diazotroph in the intertidal microbial mat system at Elkhorn Slough, CA, USA. Based on phylogenetic analyses of the 16S RNA gene, ESFC-1 appears to belong to a unique, genus-level divergence; the draft genome sequence of this strain has now been determined. Here we report features of this genome as they relate to the ecological functions and capabilities of strain ESFC-1. The 5,632,035 bp genome sequence encodes 4914 protein-coding genes and 92 RNA genes. One striking feature of this cyanobacterium is the apparent lack of either uptake or bi-directional hydrogenases typically expected within a diazotroph. Additionally, a large genomic island is found that contains numerous low GC-content genes and genes related to extracellular polysaccharide production and cell wall synthesis and maintenance.

Hydrogen (H(2)) release from photosynthetic microbial mats has contributed to the chemical evolution of Earth and could potentially be a source of renewable H(2) in the future. However, the taxonomy of H(2)-producing microorganisms (hydrogenogens) in these mats has not been previously determined. With combined biogeochemical and molecular studies of microbial mats collected from Elkhorn Slough, Monterey Bay, California, we characterized the mechanisms of H(2) production and identified a dominant hydrogenogen. Net production of H(2) was observed within the upper photosynthetic layer (0-2 mm) of the mats under dark and anoxic conditions. Pyrosequencing of rRNA gene libraries generated from this layer demonstrated the presence of 64 phyla, with Bacteriodetes, Cyanobacteria and Proteobacteria dominating the sequences. Sequencing of rRNA transcripts obtained from this layer demonstrated that Cyanobacteria dominated rRNA transcript pyrotag libraries. An OTU affiliated to Microcoleus spp. was the most abundant OTU in both rRNA gene and transcript libraries. Depriving mats of sunlight resulted in an order of magnitude decrease in subsequent nighttime H(2) production, suggesting that newly fixed carbon is critical to H(2) production. Suppression of nitrogen (N(2))-fixation in the mats did not suppress H(2) production, which indicates that co-metabolic production of H(2) during N(2)-fixation is not an important contributor to H(2) production. Concomitant production of organic acids is consistent with fermentation of recently produced photosynthate as the dominant mode of H(2) production. Analysis of rRNA % transcript:% gene ratios and H(2)-evolving bidirectional [NiFe] hydrogenase % transcript:% gene ratios indicated that Microcoelus spp. are dominant hydrogenogens in the Elkhorn Slough mats.

In the version of this Letter originally published, the name of the institute in affiliation 3 was incorrect; it read “Institute of Applied Systems Analysis” but should have read “International Institute for Applied Systems Analysis”. This has now been corrected.

An anaerobic nitrate-reducing enrichment culture was established with a cyclic saturated petroleum hydrocarbon, cyclohexane, the fate of which in anoxic environments has been scarcely investigated. GC-MS showed cyclohexylsuccinate as a metabolite, in accordance with an anaerobic enzymatic activation of cyclohexane by carbon-carbon addition to fumarate. Furthermore, long-chain cyclohexyl-substituted cell fatty acids apparently derived from cyclohexane were detected. Nitrate reduction was not only associated with cyclohexane utilization but also with striking depletion of added ammonium ions. Significantly more ammonium was consumed than could be accounted for by assimilation. This indicated the occurrence of anaerobic ammonium oxidation (anammox) with nitrite from cyclohexane-dependent nitrate reduction. Indeed, nitrite depletion was stimulated upon further addition of ammonium. Analysis of 16S rRNA genes and subsequent cell hybridization with specific probes showed that approximately 75% of the bacterial cells affiliated with the Geobacteraceae and approximately 18% with Candidatus 'Brocadia anammoxidans' (member of the Planctomycetales), an anaerobic ammonium oxidizer. These results and additional quantitative growth experiments indicated that the member of the Geobacteraceae reduced nitrate with cyclohexane to nitrite and some ammonium; the latter two and ammonium added to the medium were scavenged by anammox bacteria to yield dinitrogen. A model was established to quantify the partition of each microorganism in the overall process. Such hydrocarbon oxidation by an alleged 'denitrification' ('pseudo-denitrification'), which in reality is a dissimilatory loop through anammox, can in principle also occur in other microbial systems with nitrate-dependent hydrocarbon attenuation.

We studied microbial N 2 production via anammox and denitrification in the anoxic water column of a restored mining pit lake in Germany over an annual cycle. We obtained high-resolution hydrochemical profiles using a continuous pumping sampler. Lake Rassnitzer is permanently stratified at ca. 29 m depth, entraining anoxic water below a saline density gradient. Mixed-layer nitrate concentrations averaged ca. 200 μmol L −1 , but decreased to zero in the anoxic bottom waters. In contrast, ammonium was −1 in the mixed layer but increased in the anoxic waters to ca. 600 μmol L −1 near the sediments. In January and October, 15 N tracer measurements detected anammox activity (maximum 504 nmol N 2 L −1 d −1 in 15 NH 4 + -amended incubations), but no denitrification. In contrast, in May, N 2 production was dominated by denitrification (maximum 74 nmol N 2 L −1 d −1 ). Anammox activity in May was significantly lower than in October, as characterized by anammox rates (maximum 6 vs. 16 nmol N 2 L −1 d −1 in incubations with 15 NO 3 − ), as well as relative and absolute anammox bacterial cell abundances (0.56% vs. 0.98% of all bacteria, and 2.7×10 4 vs. 5.2×10 4 anammox cells mL −1 , respectively) (quantified by catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH) with anammox bacteria-specific probes). Anammox bacterial diversity was investigated with anammox bacteria-specific 16S rRNA gene clone libraries. The majority of anammox bacterial sequences were related to the widespread Candidatus Scalindua sorokinii/brodae cluster. However, we also found sequences related to Candidatus S. wagneri and Candidatus Brocadia fulgida, which suggests a high anammox bacterial diversity in this lake comparable with estuarine sediments.

The oxygen minimum zone (OMZ) of the Eastern Tropical South Pacific (ETSP) is 1 of the 3 major regions in the world where oceanic nitrogen is lost in the pelagic realm. The recent identification of anammox, instead of denitrification, as the likely prevalent pathway for nitrogen loss in this OMZ raises strong questions about our understanding of nitrogen cycling and organic matter remineralization in these waters. Without detectable denitrification, it is unclear how NH 4 + is remineralized from organic matter and sustains anammox or how secondary NO 2 − maxima arise within the OMZ. Here we show that in the ETSP-OMZ, anammox obtains 67% or more of NO 2 − from nitrate reduction, and 33% or less from aerobic ammonia oxidation, based on stable-isotope pairing experiments corroborated by functional gene expression analyses. Dissimilatory nitrate reduction to ammonium was detected in an open-ocean setting. It occurred throughout the OMZ and could satisfy a substantial part of the NH 4 + requirement for anammox. The remaining NH 4 + came from remineralization via nitrate reduction and probably from microaerobic respiration. Altogether, deep-sea NO 3 − accounted for only ≈50% of the nitrogen loss in the ETSP, rather than 100% as commonly assumed. Because oceanic OMZs seem to be expanding because of global climate change, it is increasingly imperative to incorporate the correct nitrogen-loss pathways in global biogeochemical models to predict more accurately how the nitrogen cycle in our future ocean may respond.

Bacterial aerobic ammonium oxidation and anaerobic ammonium oxidation (anammox) are important processes in the global nitrogen cycle. Key enzymes in both processes are the octahaem cytochrome c (OCC) proteins, hydroxylamine oxidoreductase (HAO) of aerobic ammonium-oxidizing bacteria (AOB), which catalyses the oxidation of hydroxylamine to nitrite, and hydrazine oxidoreductase (HZO) of anammox bacteria, which converts hydrazine to N(2). While the genomes of AOB encode up to three nearly identical copies of hao operons, genome analysis of Candidatus'Kuenenia stuttgartiensis' showed eight highly divergent octahaem protein coding regions as possible candidates for the HZO. Based on their phylogenetic relationship and biochemical characteristics, the sequences of these eight gene products grouped in three clusters. Degenerate primers were designed on the basis of available gene sequences with the aim to detect hao and hzo genes in various ecosystems. The hao primer pairs amplified gene fragments from 738 to 1172 bp and the hzo primer pairs amplified gene fragments from 289 to 876 bp in length, when tested on genomic DNA isolated from a variety of AOB and anammox bacteria. A selection of these primer pairs was also used successfully to amplify and analyse the hao and hzo genes in community DNA isolated from different ecosystems harbouring both AOB and anammox bacteria. We propose that OCC protein-encoding genes are suitable targets for molecular ecological studies on both aerobic and anaerobic ammonium-oxidizing bacteria.

Planctomycetes are widely distributed in marine environments, where they supposedly play a role in carbon recycling. To deepen our understanding about the ecology of this sparsely studied phylum six planctomycete fosmids from two marine upwelling systems were investigated and compared with all available planctomycete genomic sequences including the as yet unpublished near-complete genomes of Blastopirellula marina DSM 3645(T) and Planctomyces maris DSM 8797(T). High numbers of sulfatase genes (41-109) were found on all marine planctomycete genomes and on two fosmids (2). Furthermore, C1 metabolism genes otherwise only known from methanogenic Archaea and methylotrophic Proteobacteria were found on two fosmids and all planctomycete genomes, except for 'Candidatus Kuenenia stuttgartiensis'. Codon usage analysis indicated high expression levels for some of these genes. In addition, novel large families of planctomycete-specific paralogs with as yet unknown functions were identified, which are notably absent from the genome of 'Candidatus Kuenenia stuttgartiensis'. The high numbers of sulfatases in marine planctomycetes characterizes them as specialists for the initial breakdown of sulfatated heteropolysaccharides and indicate their importance for recycling carbon from these compounds. The almost ubiquitous presence of C1 metabolism genes among Planctomycetes together with codon usage analysis and information from the genomes suggest a general importance of these genes for Planctomycetes other than formaldehyde detoxification. The notable absence of these genes in Candidatus K. stuttgartiensis plus the surprising lack of almost any planctomycete-specific gene within this organism reveals an unexpected distinctiveness of anammox bacteria from all other Planctomycetes.

Fixed inorganic nitrogen compounds are important nutrients for the growth of phytoplankton in marine ecosystems and are often growth limiting. Up to 450 Tg of fixed nitrogen is lost yearly in the world's oceans (9). Until recently, this loss was fully attributed to heterotrophic denitrification. Thirty to 50% of the nitrogen loss occurs in upwelling areas and associated oxygen minimum zones (OMZ), for instance, in the Arabian Sea and the upwelling areas off the coasts of Namibia, Chile, and Peru (31). Upwelling systems are ecosystems with intense primary production in the surface waters. The decomposition of settling biomass through aerobic respiration in the water column (6, 7) leads to oxygen depletion in deeper water layers. In the Namibian upwelling system, the oxygen concentration is often less than 10 μM in deeper layers, even though surface waters are well oxygenated (>200 μM O2). These bottom waters are characterized by a strong N deficit, i.e., a decrease in the ratio of fixed inorganic N to P relative to the Redfield ratio (16:1) (49). The observed N deficit could be partly explained by the anaerobic oxidation of ammonium (“anammox”) with nitrite to N2 in the OMZ water. The first bacteria able to anaerobically oxidize ammonium were discovered in a wastewater treatment plant (39, 65) and were affiliated with a separate cluster within the order Planctomycetales (58). Subsequently, the process was also detected in sediments (13, 18, 50, 62, 64) and in stratified water bodies like Golfo Dulce (a coastal bay in Costa Rica) and the Black Sea (12, 32). Recently it was shown that anammox is mainly responsible for the loss of fixed nitrogen as N2 from the Namibian (31) and Peruvian-Chilean upwelling systems (26) (63). Most of what is known about the physiology of anammox bacteria is based on bioreactor experiments with anammox bacteria enriched at rather high ammonium concentrations from wastewater treatment plants. However, ammonium concentrations in the Namibian and Peruvian upwelling systems are often near or below the detection limit. Bioreactor studies have also shown that 1 μM O2 reversibly inhibits the anammox metabolism (59). In the Namibian OMZ, anammox activity could be detected in samples from water depths with oxygen concentrations up to 9 μM O2 (31). Preliminary results indicated particle association of anammox bacteria. These particles were believed to provide anaerobic microenvironments for the anammox bacteria at low ambient oxygen concentrations of up to 25 μM oxygen (31). The aims of this study were to further investigate the distribution and particle association of anammox bacteria in Namibian OMZ. We used comparative 16S rRNA gene analysis and catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH) to identify and localize anammox bacteria and the cooccurring microbiota. Special emphasis was placed on comparing the relative abundances of cooccurring particle-attached groups and their abundance in the free-water phase and was placed on their potential contribution to the anammox process.

Incubation experiments with 15 N-labeled compounds (NO { and NH z ) were performed during three cruises (2002, 2004, and 2005) to study the loss of inorganic N as dinitrogen gas (N2) via denitrification and anammox in the water column of the Gotland Deep (central Baltic Sea). 15 N incubations did not provide evidence for direct conversion of NO { reduction to N2 (heterotrophic denitrification) in the suboxic (O2 , 10 mmol L 21 ) sulfide-free waters. Substantial denitrification rates (up to 2.7 mmol L21 d21 N2) were measured in water samples collected from the NO { -H2S interface (redoxcline) in 2002 and in water from the sulfidic zone in 2004, which indicates chemolithotrophic denitrification as the dominant N-loss process in both years. Massive inflows of oxygenated North Sea water from 2002 to 2003 caused a complete ventilation of the Baltic Sea with high oxygen concentrations in the Gotland Deep bottom water. After the reestablishment of the redoxcline in 2004, a newly formed suboxic zone above sulfidic waters—with NO { ,N O { , and NH z at the detection limit—was observed in spring 2005. The development of this zone was associated with a several-fold increase in reduced and oxidized manganese and with a shift from denitrification to anammox as the main N-loss process. Fluorescence in situ hybridization analysis confirmed the presence of anammox bacteria and the number of anammox cells was consistent with the observed N2 production rates in 2005.

Microbial communities are essential to the function of virtually all ecosystems and eukaryotes, including humans. However, it is still a major challenge to identify microbial cells active under natural conditions in complex systems. In this study, we developed a new method to identify and sort active microbes on the single-cell level in complex samples using stable isotope probing with heavy water (D2O) combined with Raman microspectroscopy. Incorporation of D2O-derived D into the biomass of autotrophic and heterotrophic bacteria and archaea could be unambiguously detected via C-D signature peaks in single-cell Raman spectra, and the obtained labeling pattern was confirmed by nanoscaleresolution secondary ion MS. In fast-growing Escherichia coli cells, label detection was already possible after 20 min. For functional analyses of microbial communities, the detection of D incorporation from D2O in individual microbial cells via Raman microspectroscopy can be directly combined with FISH for the identification of active microbes. Applying this approach to mouse cecal microbiota revealed that the host-compound foragers Akkermansia muciniphila and Bacteroides acidifaciens exhibited distinctive response patterns to amendments of mucin and sugars. By Ramanbased cell sorting of active (deuterated) cells with optical tweezers and subsequent multiple displacement amplification and DNA sequencing, novel cecal microbes stimulated by mucin and/ or glucosamine were identified, demonstrating the potential of the nondestructive D2O-Raman approach for targeted sorting of microbial cells with defined functional properties for singlecell genomics.

Photosynthetic microbial mats are complex, stratified ecosystems in which high rates of primary production create a demand for nitrogen, met partially by N₂ fixation. Dinitrogenase reductase (nifH) genes and transcripts from Cyanobacteria and heterotrophic bacteria (for example, Deltaproteobacteria) were detected in these mats, yet their contribution to N2 fixation is poorly understood. We used a combined approach of manipulation experiments with inhibitors, nifH sequencing and single-cell isotope analysis to investigate the active diazotrophic community in intertidal microbial mats at Laguna Ojo de Liebre near Guerrero Negro, Mexico. Acetylene reduction assays with specific metabolic inhibitors suggested that both sulfate reducers and members of the Cyanobacteria contributed to N₂ fixation, whereas (15)N₂ tracer experiments at the bulk level only supported a contribution of Cyanobacteria. Cyanobacterial and nifH Cluster III (including deltaproteobacterial sulfate reducers) sequences dominated the nifH gene pool, whereas the nifH transcript pool was dominated by sequences related to Lyngbya spp. Single-cell isotope analysis of (15)N₂-incubated mat samples via high-resolution secondary ion mass spectrometry (NanoSIMS) revealed that Cyanobacteria were enriched in (15)N, with the highest enrichment being detected in Lyngbya spp. filaments (on average 4.4 at% (15)N), whereas the Deltaproteobacteria (identified by CARD-FISH) were not significantly enriched. We investigated the potential dilution effect from CARD-FISH on the isotopic composition and concluded that the dilution bias was not substantial enough to influence our conclusions. Our combined data provide evidence that members of the Cyanobacteria, especially Lyngbya spp., actively contributed to N₂ fixation in the intertidal mats, whereas support for significant N₂ fixation activity of the targeted deltaproteobacterial sulfate reducers could not be found.

The nonheterocystous filamentous cyanobacterium strain ESFC-1 has recently been isolated from a marine microbial mat system, where it was identified as belonging to a recently discovered lineage of active nitrogen-fixing microorganisms. Here, we report the draft genome sequence of this isolate. The assembly consists of 3 scaffolds and contains 5,632,035 bp with a GC content of 46.5%.

N(2) fixation is a key process in photosynthetic microbial mats to support the nitrogen demands associated with primary production. Despite its importance, groups that actively fix N(2) and contribute to the input of organic N in these ecosystems still remain largely unclear. To investigate the active diazotrophic community in microbial mats from the Elkhorn Slough estuary, Monterey Bay, CA, USA, we conducted an extensive combined approach, including biogeochemical, molecular and high-resolution secondary ion mass spectrometry (NanoSIMS) analyses. Detailed analysis of dinitrogenase reductase (nifH) transcript clone libraries from mat samples that fixed N(2) at night indicated that cyanobacterial nifH transcripts were abundant and formed a novel monophyletic lineage. Independent NanoSIMS analysis of (15)N(2)-incubated samples revealed significant incorporation of (15)N into small, non-heterocystous cyanobacterial filaments. Mat-derived enrichment cultures yielded a unicyanobacterial culture with similar filaments (named Elkhorn Slough Filamentous Cyanobacterium-1 (ESFC-1)) that contained nifH gene sequences grouping with the novel cyanobacterial lineage identified in the transcript clone libraries, displaying up to 100% amino-acid sequence identity. The 16S rRNA gene sequence recovered from this enrichment allowed for the identification of related sequences from Elkhorn Slough mats and revealed great sequence diversity in this cluster. Furthermore, by combining (15)N(2) tracer experiments, fluorescence in situ hybridization and NanoSIMS, in situ N(2) fixation activity by the novel ESFC-1 group was demonstrated, suggesting that this group may be the most active cyanobacterial diazotroph in the Elkhorn Slough mat. Pyrotag sequences affiliated with ESFC-1 were recovered from mat samples throughout 2009, demonstrating the prevalence of this group. This work illustrates that combining standard and single-cell analyses can link phylogeny and function to identify previously unknown key functional groups in complex ecosystems.

Marine sponges constitute major parts of coral reefs and deep-water communities. They often harbour high amounts of phylogenetically and physiologically diverse microbes, which are so far poorly characterized. Many of these sponges regulate their internal oxygen concentration by modulating their ventilation behaviour providing a suitable habitat for both aerobic and anaerobic microbes. In the present study, both aerobic (nitrification) and anaerobic (denitrification, anammox) microbial processes of the nitrogen cycle were quantified in the sponge Geodia barretti and possible involved microbes were identified by molecular techniques. Nitrification rates of 566 nmol N cm(-3) sponge day(-1) were obtained when monitoring the production of nitrite and nitrate. In support of this finding, ammonia-oxidizing Archaea (crenarchaeotes) were found by amplification of the amoA gene, and nitrite-oxidizing bacteria of the genus Nitrospira were detected based on rRNA gene analyses. Incubation experiments with stable isotopes ((15)NO(3)(-) and (15)NH(4)(+)) revealed denitrification and anaerobic ammonium oxidation (anammox) rates of 92 nmol N cm(-3) sponge day(-1) and 3 nmol N cm(-3) sponge day(-1) respectively. Accordingly, sequences closely related to 'Candidatus Scalindua sorokinii' and 'Candidatus Scalindua brodae' were detected in 16S rRNA gene libraries. The amplification of the nirS gene revealed the presence of denitrifiers, likely belonging to the Betaproteobacteria. This is the first proof of anammox and denitrification in the same animal host, and the first proof of anammox and denitrification in sponges. The close and complex interactions of aerobic, anaerobic, autotrophic and heterotrophic microbial processes are fuelled by metabolic waste products of the sponge host, and enable efficient utilization and recirculation of nutrients within the sponge-microbe system. Since denitrification and anammox remove inorganic nitrogen from the environment, sponges may function as so far unrecognized nitrogen sinks in the ocean. In certain marine environments with high sponge cover, sponge-mediated nitrogen mineralization processes might even be more important than sediment processes.