Publications in peer reviewed journals

12 Publications found
  • The membrane lipid composition of the moderately thermophilic ammonia-oxidizing archaeon Ca. Nitrosotenuis uzonensis at different growth temperatures

    Bale NJ, Palatinszky M, Rijpstra WIC, Herbold CW, Wagner M, Damste JSS
    2019 - Appl Environ Microbiol, in press
    AOA N. uzonensis lipids


    Ca. Nitrosotenuis uzonensis is the only cultured moderately thermophilic member of the thaumarchaeotal order Nitrosopumilales (NP) that contains many mesophilic marine strains. We examined its membrane lipid composition at different growth temperatures (37, 46 and 50 °C). Its lipids were all membrane-spanning glycerol dialkyl glycerol tetraethers (GDGTs), with 0 to 4 cyclopentane moieties. Crenarchaeol (cren), the characteristic thaumarchaeotal GDGT, and its isomer (crenʹ) were present in high abundance (30-70 %). The GDGT polar headgroups were mono-, di- and trihexoses and hexose/phosphohexose. The ratio of glycolipid to phospholipid GDGTs was highest in the cultures grown at 50 °C. With increasing growth temperature, the relative contribution of cren and crenʹ increased, while GDGT-0 to GDGT-4 (including isomers) decreased. TEX86 (tetraether index of tetraethers consisting of 86 carbons)-derived temperatures were much lower than the actual growth temperatures, further demonstrating that TEX86 does not accurately reflect the membrane lipid adaptation of thermophilic Thaumarchaeota. As temperature increased, specific GDGTs changed relative to their isomers, possibly representing temperature adaption-induced changes in cyclopentane ring stereochemistry. Comparison of a wide range of thaumarcheotal core lipid compositions revealed the Ca. N. uzonensis cultures clustered separately fromother members of the NP order and the Nitrososphaerales (NS) order. While phylogeny generally seems to have a strong influence on GDGT distribution, our analysis of Ca. Nitrosotenuis uzonensis demonstrates that its terrestrial, higher temperature niche has led to a lipid composition that clearly differentiates it from other NP members and that this difference is mostly driven by its high crenʹ content.

  • Proteomic response of three marine ammonia-oxidizing archaea to hydrogen peroxide and their metabolic interactions with a heterotrophic alphaproteobacterium

    Bayer B, Pelikan C, Bittner MJ, Reinthaler T, Könneke M, Herndl GJ, Offre P
    2019 - mSystems, 4: e00181-1


    Ammonia-oxidizing archaea (AOA) play an important role in the nitrogen cycle and account for a considerable fraction of the prokaryotic plankton in the ocean. Most AOA lack the hydrogen peroxide (H2O2)-detoxifying enzyme catalase, and some AOA have been shown to grow poorly under conditions of exposure to H2O2. However, differences in the degrees of H2O2 sensitivity of different AOA strains, the physiological status of AOA cells exposed to H2O2, and their molecular response to H2O2 remain poorly characterized. Further, AOA might rely on heterotrophic bacteria to detoxify H2O2, and yet the extent and variety of costs and benefits involved in these interactions remain unclear. Here, we used a proteomics approach to compare the protein profiles of three Nitrosopumilus strains grown in the presence and absence of catalase and in coculture with the heterotrophic alphaproteobacterium Oceanicaulis alexandrii. We observed that most proteins detected at a higher relative abundance in H2O2-exposed Nitrosopumilus cells had no known function in oxidative stress defense. Instead, these proteins were putatively involved in the remodeling of the extracellular matrix, which we hypothesize to be a strategy limiting the influx of H2O2 into the cells. Using RNA-stable isotope probing, we confirmed that O. alexandrii cells growing in coculture with the Nitrosopumilus strains assimilated Nitrosopumilus-derived organic carbon, suggesting that AOA could recruit H2O2-detoxifying bacteria through the release of labile organic matter. Our results contribute new insights into the response of AOA to H2O2 and highlight the potential ecological importance of their interactions with heterotrophic free-living bacteria in marine environments.

  • Expansion of Thaumarchaeota habitat range is correlated with horizontal transfer of ATPase operons

    Wang B, Qin W, Ren Y, Zhou X, Young Jung M, Han P, Eloe-Fadrosh EA, Li M, Zheng Y, Lu L, Yan X, Ji J, Liu Y, Liu L, Heiner C, Hall R, Martens-Habbena W, Herbold CW, Rhee S-K, Bartlett DH, Huang L, Ingalls AE, Wagner M, Stahl DA, Jia Z
    2019 - ISME J, in press
    Phylogenetic ATPase tree of Thaumarchaeota


    Thaumarchaeota are responsible for a significant fraction of ammonia oxidation in the oceans and in soils that range from alkaline to acidic. However, the adaptive mechanisms underpinning their habitat expansion remain poorly understood. Here we show that expansion into acidic soils and the high pressures of the hadopelagic zone of the oceans is tightly linked to the acquisition of a variant of the energy-yielding ATPases via horizontal transfer. Whereas the ATPase genealogy of neutrophilic Thaumarchaeota is congruent with their organismal genealogy inferred from concatenated conserved proteins, a common clade of V-type ATPases unites phylogenetically distinct clades of acidophilic/acid-tolerant and piezophilic/piezotolerant species. A presumptive function of pumping cytoplasmic protons at low pH is consistent with the experimentally observed increased expression of the V-ATPase in an acid-tolerant thaumarchaeote at low pH. Consistently, heterologous expression of the thaumarchaeal V-ATPase significantly increased the growth rate of E. coli at low pH. Its adaptive significance to growth in ocean trenches may relate to pressure-related changes in membrane structure in which this complex molecular machine must function. Together, our findings reveal that the habitat expansion of Thaumarchaeotais tightly correlated with extensive horizontal transfer of atp operons.

  • A multicolor fluorescence in situ hybridization approach using an extended set of fluorophores to visualize microorganisms

    Lukumbuzya M, Schmid M, Pjevac P, Daims H
    2019 - Front Microbiol, 10: 1383


    Fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes is a key method for the detection of (uncultured) microorganisms in environmental and medical samples. A major limitation of standard FISH protocols, however, is the small number of phylogenetically distinct target organisms that can be detected simultaneously. In this study, we introduce a multicolor FISH approach that uses eight fluorophores with distinct spectral properties, which can unambiguously be distinguished by confocal laser scanning microscopy combined with white light laser technology. Hybridization of rRNA-targeted DNA oligonucleotide probes, which were mono-labeled with these fluorophores, to Escherichia coli cultures confirmed that the fluorophores did not affect probe melting behavior. Application of the new multicolor FISH method enabled the differentiation of seven (potentially up to eight) phylogenetically distinct microbial populations in an artificial community of mixed pure cultures (five bacteria, one archaeon, and one yeast strain) and in activated sludge from a full-scale wastewater treatment plant. In contrast to previously published multicolor FISH approaches, this method does not rely on combinatorial labeling of the same microorganisms with different fluorophores, which is prone to biases. Furthermore, images acquired by this method do not require elaborate post-processing prior to analysis. We also demonstrate that the newly developed multicolor FISH method is compatible with an improved cell fixation protocol for FISH targeting Gram-negative bacterial populations. This fixation approach uses agarose embedding during formaldehyde fixation to better preserve the three-dimensional structure of spatially complex samples such as biofilms and activated sludge flocs. The new multicolor FISH approach should be highly suitable for studying structural and functional aspects of microbial communities in virtually all types of samples that can be analyzed by conventional FISH methods.

  • Specific Micropollutant Biotransformation Pattern by the Comammox Bacterium .

    Han P, Yu Y, Zhou L, Tian Z, Li Z, Hou L, Liu M, Wu Q, Wagner M, Men Y
    2019 - Environ. Sci. Technol., in press
    Comammox biodegradation graphical abstract


    The recently discovered complete ammonia-oxidizing (comammox) bacteria occur in various environments, including wastewater treatment plants. To better understand their role in micropollutant biotransformation in comparison with ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA), we investigated the biotransformation capability of (the only comammox isolate) for 17 micropollutants. Asulam, fenhexamid, mianserin, and ranitidine were biotransformed by , (AOA), and Nm90 (AOB). More distinctively, carbendazim, a benzimidazole fungicide, was exclusively biotransformed by . The biotransformation of carbendazim only occurred when was supplied with ammonia but not nitrite as the energy source. The exclusive biotransformation of carbendazim by was likely enabled by an enhanced substrate promiscuity of its unique AMO and its much higher substrate (for ammonia) affinity compared with the other two ammonia oxidizers. One major plausible transformation product (TP) of carbendazim is a hydroxylated form at the aromatic ring, which is consistent with the function of AMO. These findings provide fundamental knowledge on the micropollutant degradation potential of a comammox bacterium to better understand the fate of micropollutants in nitrifying environments.

  • Characterization of a thaumarchaeal symbiont that drives incomplete nitrification in the tropical sponge Ianthella basta.

    Moeller FU, Webster NS, Herbold CW, Behnam F, Domman D, Albertsen M, Mooshammer M, Markert S, Turaev D, Becher D, Rattei T, Schweder T, Richter A, Watzka M, Nielsen PH, Wagner M
    2019 - Environ. Microbiol., in press
    Metabolic scheme of the AOA symbiont in I. basta


    Marine sponges represent one of the few eukaryotic groups that frequently harbour symbiotic members of the Thaumarchaeota, which are important chemoautotrophic ammonia-oxidizers in many environments. However, in most studies, direct demonstration of ammonia-oxidation by these archaea within sponges is lacking, and little is known about sponge-specific adaptations of ammonia-oxidizing archaea (AOA). Here, we characterized the thaumarchaeal symbiont of the marine sponge Ianthella basta using metaproteogenomics, fluorescence in situ hybridization, qPCR and isotope-based functional assays. 'Candidatus Nitrosospongia ianthellae' is only distantly related to cultured AOA. It is an abundant symbiont that is solely responsible for nitrite formation from ammonia in I. basta that surprisingly does not harbour nitrite-oxidizing microbes. Furthermore, this AOA is equipped with an expanded set of extracellular subtilisin-like proteases, a metalloprotease unique among archaea, as well as a putative branched-chain amino acid ABC transporter. This repertoire is strongly indicative of a mixotrophic lifestyle and is (with slight variations) also found in other sponge-associated, but not in free-living AOA. We predict that this feature as well as an expanded and unique set of secreted serpins (protease inhibitors), a unique array of eukaryotic-like proteins, and a DNA-phosporothioation system, represent important adaptations of AOA to life within these ancient filter-feeding animals.

  • Indications for enzymatic denitrification to NO at low pH in an ammonia-oxidizing archaeon.

    Jung MY, Gwak JH, Rohe L, Giesemann A, Kim JG, Well R, Madsen EL, Herbold CW, Wagner M, Rhee SK
    2019 - ISME J, in press
    P450 NOR in AOA


    Nitrous oxide (NO) is a key climate change gas and nitrifying microbes living in terrestrial ecosystems contribute significantly to its formation. Many soils are acidic and global change will cause acidification of aquatic and terrestrial ecosystems, but the effect of decreasing pH on NO formation by nitrifiers is poorly understood. Here, we used isotope-ratio mass spectrometry to investigate the effect of acidification on production of NO by pure cultures of two ammonia-oxidizing archaea (AOA; Nitrosocosmicus oleophilus and Nitrosotenuis chungbukensis) and an ammonia-oxidizing bacterium (AOB; Nitrosomonas europaea). For all three strains acidification led to increased emission of NO. However, changes of N site preference (SP) values within the NO molecule (as indicators of pathways for NO formation), caused by decreasing pH, were highly different between the tested AOA and AOB. While acidification decreased the SP value in the AOB strain, SP values increased to a maximum value of 29‰ in N. oleophilus. In addition, N-nitrite tracer experiments showed that acidification boosted nitrite transformation into NO in all strains, but the incorporation rate was different for each ammonia oxidizer. Unexpectedly, for N. oleophilus more than 50% of the NO produced at pH 5.5 had both nitrogen atoms from nitrite and we demonstrated that under these conditions expression of a putative cytochrome P450 NO reductase is strongly upregulated. Collectively, our results indicate that N. oleophilus might be able to enzymatically denitrify nitrite to NO at low pH.

  • Cometabolic biotransformation and microbial-mediated abiotic transformation of sulfonamides by three ammonia oxidizers.

    Zhou LJ, Han P, Yu Y, Wang B, Men Y, Wagner M, Wu QL
    2019 - Water Res., 444-453


    The abilities of three phylogenetically distant ammonia oxidizers, Nitrososphaera gargensis, an ammonia-oxidizing archaeon (AOA); Nitrosomomas nitrosa Nm90, an ammonia-oxidizing bacterium (AOB); and Nitrospira inopinata, the only complete ammonia oxidizer (comammox) available as a pure culture, to biotransform seven sulfonamides (SAs) were investigated. The removals and protein-normalized biotransformation rate constants indicated that the AOA strain N. gargensis exhibited the highest SA biotransformation rates, followed by N. inopinata and N. nitrosa Nm90. The transformation products (TPs) of sulfadiazine (SDZ), sulfamethazine (SMZ) and sulfamethoxazole (SMX) and the biotransformation mechanisms were evaluated. Based on the analysis of the TP formulas and approximate structures, it was found that during biotransformation, i) the AOA strain carried out SA deamination, hydroxylation, and nitration; ii) the AOB strain mainly performed SA deamination; and iii) the comammox isolate participated only in deamination reactions. It is proposed that deamination was catalyzed by deaminases while hydroxylation and nitration were mediated by nonspecific activities of the ammonia monooxygenase (AMO). Additionally, it was demonstrated that among the three ammonia oxidizers, only AOB contributed to the formation of pterin-SA conjugates. The biotransformation of SDZ, SMZ and SMX occurred only when ammonia oxidation was active, suggesting a cometabolic transformation mechanism. Interestingly, SAs could also be transformed by hydroxylamine, an intermediate of ammonia oxidation, suggesting that in addition to enzymatic conversions, a microbially induced abiotic mechanism contributes to SA transformation during ammonia oxidation. Overall, using experiments with pure cultures, this study provides important insights into the roles played by ammonia oxidizers in SA biotransformation.

  • Low yield and abiotic origin of NO formed by the complete nitrifier Nitrospira inopinata.

    Kits KD, Jung MY, Vierheilig J, Pjevac P, Sedlacek CJ, Liu S, Herbold C, Stein LY, Richter A, Wissel H, Brüggemann N, Wagner M, Daims H
    2019 - Nat Commun, 1: 1836
    Nitrous oxide comammox


    Nitrous oxide (NO) and nitric oxide (NO) are atmospheric trace gases that contribute to climate change and affect stratospheric and ground-level ozone concentrations. Ammonia oxidizing bacteria (AOB) and archaea (AOA) are key players in the nitrogen cycle and major producers of NO and NO globally. However, nothing is known about NO and NO production by the recently discovered and widely distributed complete ammonia oxidizers (comammox). Here, we show that the comammox bacterium Nitrospira inopinata is sensitive to inhibition by an NO scavenger, cannot denitrify to NO, and emits NO at levels that are comparable to AOA but much lower than AOB. Furthermore, we demonstrate that NO formed by N. inopinata formed under varying oxygen regimes originates from abiotic conversion of hydroxylamine. Our findings indicate that comammox microbes may produce less NO during nitrification than AOB.

  • Mucispirillum schaedleri antagonizes Salmonella virulence to protect mice against colitis

    Herp S, Brugiroux S, Garzetti D, Ring D, Jochum LM, Beutler M, Eberl C, Hussain S, Walter S, Gerlach RG, Ruscheweyh HJ, Huson D, Sellin ME, Slack E, Hanson B, Loy A, Baines JF, Rausch P, Basic M, Bleich A, Berry D, Stecher B
    2019 - Cell Host Microbe, 25: 681-694


    The microbiota and the gastrointestinal mucus layer play a pivotal role in protection against non-typhoidal Salmonellaenterica serovar Typhimurium (S. Tm) colitis. Here, we analyzed the course of Salmonella colitis in mice lacking a functional mucus layer in the gut. Unexpectedly, in contrast to mucus-proficient littermates, genetically deficient mice were protected against Salmonella-induced gut inflammation in the streptomycin colitis model. This correlated with microbiota alterations and enrichment of the bacterial phylum Deferribacteres. Using gnotobiotic mice associated with defined bacterial consortia, we causally linked Mucispirillum schaedleri, currently the sole known representative of Deferribacteres present in the mammalian microbiota, to host protection against S. Tm colitis. Inhibition by M. schaedleri involves interference with S. Tm invasion gene expression, partly by competing for anaerobic electron acceptors. In conclusion, this study establishes M. schaedleri, a core member of the murine gut microbiota, as a key antagonist of S. Tm virulence in the gut.

  • Rapid Transfer of Plant Photosynthates to Soil Bacteria via Ectomycorrhizal Hyphae and Its Interaction With Nitrogen Availability.

    Gorka S, Dietrich M, Mayerhofer W, Gabriel R, Wiesenbauer J, Martin V, Zheng Q, Imai B, Prommer J, Weidinger M, Schweiger P, Eichorst SA, Wagner M, Richter A, Schintlmeister A, Woebken D, Kaiser C
    2019 - Front Microbiol, 168


    Plant roots release recent photosynthates into the rhizosphere, accelerating decomposition of organic matter by saprotrophic soil microbes ("rhizosphere priming effect") which consequently increases nutrient availability for plants. However, about 90% of all higher plant species are mycorrhizal, transferring a significant fraction of their photosynthates directly to their fungal partners. Whether mycorrhizal fungi pass on plant-derived carbon (C) to bacteria in root-distant soil areas, i.e., incite a "hyphosphere priming effect," is not known. Experimental evidence for C transfer from mycorrhizal hyphae to soil bacteria is limited, especially for ectomycorrhizal systems. As ectomycorrhizal fungi possess enzymatic capabilities to degrade organic matter themselves, it remains unclear whether they cooperate with soil bacteria by providing photosynthates, or compete for available nutrients. To investigate a possible C transfer from ectomycorrhizal hyphae to soil bacteria, and its response to changing nutrient availability, we planted young beech trees () into "split-root" boxes, dividing their root systems into two disconnected soil compartments. Each of these compartments was separated from a litter compartment by a mesh penetrable for fungal hyphae, but not for roots. Plants were exposed to a C-CO-labeled atmosphere, while N-labeled ammonium and amino acids were added to one side of the split-root system. We found a rapid transfer of recent photosynthates via ectomycorrhizal hyphae to bacteria in root-distant soil areas. Fungal and bacterial phospholipid fatty acid (PLFA) biomarkers were significantly enriched in hyphae-exclusive compartments 24 h after C-CO-labeling. Isotope imaging with nanometer-scale secondary ion mass spectrometry (NanoSIMS) allowed for the first time visualization of plant-derived C and N taken up by an extraradical fungal hypha, and in microbial cells thriving on hyphal surfaces. When N was added to the litter compartments, bacterial biomass, and the amount of incorporated C strongly declined. Interestingly, this effect was also observed in adjacent soil compartments where added N was only available for bacteria through hyphal transport, indicating that ectomycorrhizal fungi were acting on soil bacteria. Together, our results demonstrate that (i) ectomycorrhizal hyphae rapidly transfer plant-derived C to bacterial communities in root-distant areas, and (ii) this transfer promptly responds to changing soil nutrient conditions.

  • Cyanate and urea are substrates for nitrification by Thaumarchaeota in the marine environment.

    Kitzinger K, Padilla CC, Marchant HK, Hach PF, Herbold CW, Kidane AT, Könneke M, Littmann S, Mooshammer M, Niggemann J, Petrov S, Richter A, Stewart FJ, Wagner M, Kuypers MMM, Bristow LA
    2019 - Nat Microbiol, 2: 234-243
    Cyanate use by thaumarchaeota


    Ammonia-oxidizing archaea of the phylum Thaumarchaeota are among the most abundant marine microorganisms. These organisms thrive in the oceans despite ammonium being present at low nanomolar concentrations. Some Thaumarchaeota isolates have been shown to utilize urea and cyanate as energy and N sources through intracellular conversion to ammonium. Yet, it is unclear whether patterns observed in culture extend to marine Thaumarchaeota, and whether Thaumarchaeota in the ocean directly utilize urea and cyanate or rely on co-occurring microorganisms to break these substrates down to ammonium. Urea utilization has been reported for marine ammonia-oxidizing communities, but no evidence of cyanate utilization exists for marine ammonia oxidizers. Here, we demonstrate that in the Gulf of Mexico, Thaumarchaeota use urea and cyanate both directly and indirectly as energy and N sources. We observed substantial and linear rates of nitrite production from urea and cyanate additions, which often persisted even when ammonium was added to micromolar concentrations. Furthermore, single-cell analysis revealed that the Thaumarchaeota incorporated ammonium-, urea- and cyanate-derived N at significantly higher rates than most other microorganisms. Yet, no cyanases were detected in thaumarchaeal genomic data from the Gulf of Mexico. Therefore, we tested cyanate utilization in Nitrosopumilus maritimus, which also lacks a canonical cyanase, and showed that cyanate was oxidized to nitrite. Our findings demonstrate that marine Thaumarchaeota can use urea and cyanate as both an energy and N source. On the basis of these results, we hypothesize that urea and cyanate are substrates for ammonia-oxidizing Thaumarchaeota throughout the ocean.

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