Publications

Publications in peer reviewed journals

8 Publications found
  • Evaluation of primers targeting the diazotroph functional gene and development of NifMAP – a bioinformatics pipeline for analyzing nifH amplicon data

    Angel R, Nepel M, Panhölzl C, Schmidt H, Herbold CW, Eichorst SA, Woebken D
    2018 - Front Microbiol, 9: 1-15

    Abstract: 

    Diazotrophic microorganisms introduce biologically available nitrogen (N) to the global N cycle through the activity of the nitrogenase enzyme. The genetically conserved dinitrogenase reductase (nifH) gene is phylogenetically distributed across four clusters (I-IV) and is widely used as a marker gene for N2 fixation, permitting investigators to study the genetic diversity of diazotrophs in nature and target potential participants in N2 fixation. To date there have been limited, standardized pipelines for the nifH functional gene, which is in stark contrast to the rRNA gene. Here we present a bioinformatics pipeline for processing nifH amplicon datasets – NifMAP (“NifH MiSeq Illumina amplicon Analysis Pipeline”), which as a novel aspect uses Hidden-Markov models to filter out homologous genes to nifH. By using this pipeline, we evaluated the broadly inclusive primer pairs (Ueda19F-R6, IGK3-DVV, F2-R6) that target the nifH gene. To evaluate any systematic biases, the nifH gene was amplified with the aforementioned primer pairs in a diverse collection of environmental samples (soils, rhizosphere and roots samples, biological soil crusts and estuarine samples), in addition to a nifH mock community consisting of six phylogenetically diverse members. We noted that all primer pairs co-amplified nifH homologs to varying degrees; up to 90% of the amplicons were nifH homologs with IGK3-DVV in some samples (rhizosphere and roots from tall oat-grass). In regards to specificity, we observed some degree of bias across the primer pairs. For example, primer pair F2-R6 discriminated against cyanobacteria (amongst others), yet captured many sequences from subclusters IIIE and IIIL-N. These aforementioned subclusters were largely missing by the primer pair IGK3-DVV, which also tended to discriminate against Alphaproteobacteria, but amplified sequences within clusters IIIC (affiliated with Clostridia) and clusters IVB and IVC. Primer pair Ueda19F-R6 exhibited the least bias and successfully captured diazotrophs in cluster I and subclusters IIIE, IIIL, IIIM and IIIN, but discriminated against Firmicutes and subcluster IIIC. Taken together, our newly established bioinformatics pipeline, NifMAP, along with our systematic evaluations of nifH primer pairs permit more robust, high-throughput investigations of diazotrophs in diverse environments. 

  • Application of stable-isotope labelling techniques for the detection of active diazotrophs

    Angel R, Panhölzl C, Gabriel R, Herbold CW, Wanek W, Richter A, Eichorst SA, Woebken D
    2018 - Environmental Microbiology, 20: 44-61

    Abstract: 

    Investigating active participants in the fixation of dinitrogen gas is vital as N is often a limiting factor for primary production. Biological nitrogen fixation (BNF) is performed by a diverse guild of bacteria and archaea (diazotrophs), which can be free-living or symbionts. Free-living diazotrophs are widely distributed in the environment, yet our knowledge about their identity and ecophysiology is still limited. A major challenge in investigating this guild is inferring activity from genetic data as this process is highly regulated. To address this challenge, we evaluated and improved several 15N-based methods for detecting N2 fixation activity (with a focus on soil samples) and studying active diazotrophs. We compared the acetylene reduction assay and the 15N2 tracer method and demonstrated that the latter is more sensitive in samples with low activity. Additionally, tracing 15N into microbial RNA provides much higher sensitivity compared to bulk soil analysis. Active soil diazotrophs were identified with a 15N-RNA-SIP approach optimized for environmental samples and benchmarked to 15N-DNA-SIP. Lastly, we investigated the feasibility of using SIP-Raman microspectroscopy for detecting 15N-labelled cells. Taken together, these tools allow identifying and investigating active free-living diazotrophs in a highly sensitive manner in diverse environments, from bulk to the single-cell level.

  • 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
    2018 - Nature Microbiology, in press
    Cyanate use by thaumarchaeota

    Abstract: 

    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.

  • The draft genome sequence of “Nitrospira lenta” strain BS10, a nitrite oxidizing bacterium isolated from activated sludge

    Sakoula D, Nowka B, Spieck E, Daims H, Lücker S
    2018 - Stand Genomic Sci, 13: 32

    Abstract: 

    The genus Nitrospira is considered to be the most widespread and abundant group of nitrite-oxidizing bacteria in many natural and man-made ecosystems. However, the ecophysiological versatility within this phylogenetic group remains highly understudied, mainly due to the lack of pure cultures and genomic data. To further expand our understanding of this biotechnologically important genus, we analyzed the high quality draft genome of “Nitrospira lenta” strain BS10, a sublineage II Nitrospira that was isolated from a municipal wastewater treatment plant in Hamburg, Germany. The genome of “N. lenta” has a size of 3,756,190 bp and contains 3968 genomic objects, of which 3907 are predicted protein-coding sequences. Thorough genome annotation allowed the reconstruction of the “N. lenta” core metabolism for energy conservation and carbon fixation. Comparative analyses indicated that most metabolic features are shared with N. moscoviensis and “N. defluvii”, despite their ecological niche differentiation and phylogenetic distance. In conclusion, the genome of “N. lenta” provides important insights into the genomic diversity of the genus Nitrospira and provides a foundation for future comparative genomic studies that will generate a better understanding of the nitrification process.

  • Microbial nitrogen limitation in the mammalian large intestine.

    Reese AT, Pereira FC, Schintlmeister A, Berry D, Wagner M, Hale LP, Wu A, Jiang S, Durand HK, Zhou X, Premont RT, Diehl AM, O'Connell TM, Alberts SC, Kartzinel TR, Pringle RM, Dunn RR, Wright JP, David LA
    2018 - Nat Microbiol, in press

    Abstract: 

    Resource limitation is a fundamental factor governing the composition and function of ecological communities. However, the role of resource supply in structuring the intestinal microbiome has not been established and represents a challenge for mammals that rely on microbial symbionts for digestion: too little supply might starve the microbiome while too much might starve the host. We present evidence that microbiota occupy a habitat that is limited in total nitrogen supply within the large intestines of 30 mammal species. Lowering dietary protein levels in mice reduced their faecal concentrations of bacteria. A gradient of stoichiometry along the length of the gut was consistent with the hypothesis that intestinal nitrogen limitation results from host absorption of dietary nutrients. Nitrogen availability is also likely to be shaped by host-microbe interactions: levels of host-secreted nitrogen were altered in germ-free mice and when bacterial loads were reduced via experimental antibiotic treatment. Single-cell spectrometry revealed that members of the phylum Bacteroidetes consumed nitrogen in the large intestine more readily than other commensal taxa did. Our findings support a model where nitrogen limitation arises from preferential host use of dietary nutrients. We speculate that this resource limitation could enable hosts to regulate microbial communities in the large intestine. Commensal microbiota may have adapted to nitrogen-limited settings, suggesting one reason why excess dietary protein has been associated with degraded gut-microbial ecosystems.

  • Ammonia Monooxygenase-Mediated Cometabolic Biotransformation and Hydroxylamine-Mediated Abiotic Transformation of Micropollutants in an AOB/NOB Coculture.

    Yu Y, Han P, Zhou LJ, Li Z, Wagner M, Men Y
    2018 - Environ. Sci. Technol., 16: 9196-9205

    Abstract: 

    Biotransformation of various micropollutants (MPs) has been found to be positively correlated with nitrification in activated sludge communities. To further elucidate the roles played by ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB), we investigated the biotransformation capabilities of an NOB pure culture ( Nitrobacter sp.) and an AOB ( Nitrosomonas europaea)/NOB ( Nitrobacter sp.) coculture for 15 MPs, whose biotransformation was reported previously to be associated with nitrification. The NOB pure culture did not biotransform any investigated MP, whereas the AOB/NOB coculture was capable of biotransforming six MPs (i.e., asulam, bezafibrate, fenhexamid, furosemide, indomethacin, and rufinamide). Transformation products (TPs) were identified, and tentative structures were proposed. Inhibition studies with octyne, an ammonia monooxygenase (AMO) inhibitor, suggested that AMO was the responsible enzyme for MP transformation that occurred cometabolically. For the first time, hydroxylamine, a key intermediate of all aerobic ammonia oxidizers, was found to react with several MPs at concentrations typically occurring in AOB batch cultures. All of these MPs were also biotransformed by the AOB/NOB coculture. Moreover, the same asulam TPs were detected in both biotransformation and hydroxylamine-treated abiotic transformation experiments, whereas rufinamide TPs formed from biological transformation were not detected during hydroxylamine-mediated abiotic transformation, which was consistent with the inability of rufinamide abiotic transformation by hydroxylamine. Thus, in addition to cometabolism likely carried out by AMO, an abiotic transformation route indirectly mediated by AMO might also contribute to MP biotransformation by AOB.

  • Characterization of the first “Candidatus Nitrotoga” isolate reveals metabolic versatility and separate evolution of widespread nitrite-oxidizing bacteria

    Kitzinger K, Koch H, Lücker S, Sedlacek CJ, Herbold CW, Schwarz J, Daebeler A, Mueller AJ, Lukumbuzya M, Romano S, Leisch N, Karst SM, Kirkegaard R, Albertsen M, Nielsen PH, Wagner M, Daims H
    2018 - mBio, 9: e01186-18

    Abstract: 

    Nitrification is a key process of the biogeochemical nitrogen cycle and of biological wastewater treatment. The second step, nitrite oxidation to nitrate, is catalyzed by phylogenetically diverse, chemolithoautotrophic nitrite-oxidizing bacteria (NOB). Uncultured NOB from the genus “Candidatus Nitrotoga” are widespread in natural and engineered ecosystems. Knowledge about their biology is sparse, because no genomic information and no pure “Ca. Nitrotoga” culture was available. Here we obtained the first “Ca. Nitrotoga” isolate from activated sludge. This organism, “Candidatus Nitrotoga fabula,” prefers higher temperatures (>20°C; optimum, 24 to 28°C) than previous “Ca. Nitrotoga” enrichments, which were described as cold-adapted NOB. “Ca. Nitrotoga fabula” also showed an unusually high tolerance to nitrite (activity at 30 mM NO2) and nitrate (up to 25 mM NO3). Nitrite oxidation followed Michaelis-Menten kinetics, with an apparent Km (Km(app)) of ~89 µM nitrite and a Vmax of ~28 µmol of nitrite per mg of protein per h. Key metabolic pathways of “Ca. Nitrotoga fabula” were reconstructed from the closed genome. “Ca. Nitrotoga fabula” possesses a new type of periplasmic nitrite oxidoreductase belonging to a lineage of mostly uncharacterized proteins. This novel enzyme indicates (i) separate evolution of nitrite oxidation in “Ca. Nitrotoga” and other NOB, (ii) the possible existence of phylogenetically diverse, unrecognized NOB, and (iii) together with new metagenomic data, the potential existence of nitrite-oxidizing archaea. For carbon fixation, “Ca. Nitrotoga fabula” uses the Calvin-Benson-Bassham cycle. It also carries genes encoding complete pathways for hydrogen and sulfite oxidation, suggesting that alternative energy metabolisms enable “Ca. Nitrotoga fabula” to survive nitrite depletion and colonize new niches.

  • Cultivation and genomic analysis of “Candidatus Nitrosocaldus islandicus”, an obligately thermophilic, ammonia-oxidizing thaumarchaeon from a hot spring biofilm in Graendalur valley, Iceland

    Daebeler A, Herbold CW, Vierheilig J, Sedlacek CJ, Pjevac P, Albertsen M, Kirkegaard RH, De La Torre JR, Daims H, Wagner M
    2018 - Front Microbiol, 9: 193

    Abstract: 

    Ammonia-oxidizing archaea (AOA) within the phylum Thaumarchaeota are the only known aerobic ammonia oxidizers in geothermal environments. Although molecular data indicate the presence of phylogenetically diverse AOA from the Nitrosocaldus clade, group 1.1b and group 1.1a Thaumarchaeota in terrestrial high-temperature habitats, only one enrichment culture of an AOA thriving above 50 °C has been reported and functionally analyzed. In this study, we physiologically and genomically characterized a newly discovered thaumarchaeon from the deep-branching Nitrosocaldaceae family of which we have obtained a high (~85 %) enrichment from biofilm of an Icelandic hot spring (73 °C). This AOA, which we provisionally refer to as “Candidatus Nitrosocaldus islandicus”, is an obligately thermophilic, aerobic chemolithoautotrophic ammonia oxidizer, which stoichiometricall converts ammonia to nitrite at temperatures between 50 °C and 70 °C. “Ca. N. islandicus” encodes the expected repertoire of enzymes proposed to be required for archaeal ammonia oxidation, but unexpectedly lacks a nirK gene and also possesses no identifiable other enzyme for nitric oxide (NO) generation*. Nevertheless, ammonia oxidation by this AOA appears to be NO-dependent as “Ca. N. islandicus” is, like all other tested AOA, inhibited by the addition of an NO scavenger. Furthermore, comparative genomics revealed that “Ca. N. islandicus” has the potential for aromatic amino acid fermentation as its genome encodes an indolepyruvate oxidoreductase (iorAB) as well as a type 3b hydrogenase, which are not present in any other sequenced AOA. A further surprising genomic feature of this thermophilic ammonia oxidizer is the absence of DNA polymerase D genes – one of the predominant replicative DNA polymerases in all other ammonia-oxidizing Thaumarchaeota. Collectively, our findings suggest that metabolic versatility and DNA replication might differ substantially between obligately thermophilic and other AOA.

Book chapters and other publications

2 Publications found
  • Stickstoffkreisläufe in der Abwasserreinigung - neue und bewährte Wege

    2018 - 31-46. in Wiener Mitteilungen Wasser-Abwasser-Gewässer, vol. 247. (Krampe, J; Kreuzinger, N)

    Abstract: 

    Der klassische Weg zur Stickstoff-Eliminierung in Kläranlagen beruht auf der Kombination von Nitrifikation und Denitrifikation. In den letzten Jahren haben molekularbiologische Methoden eine Vielzahl neuer Einblicke in die Biologie der Nitrifikanten (Ammoniak- und Nitritoxidierer) ergeben. Diese Erkenntnisse beinhalten eine unerwartet hohe Diversität dieser Bakterien, alternative Stoffwechselwege sowie komplett allein nitrifizierende Mikroben (Comammox-Organismen). Das resultierende neue Bild der Nitrifikation weicht stark vom etablierten Lehrbuchwissen ab. Ein kosten- und energieeffizienterer Weg zur Stickstoff-Eliminierung nutzt die anaerobe Ammoniumoxidation (Anammox-Prozess). Dieser Ansatz nutzt extrem langsam wachsende Bakterien und erfordert die selektive Unterdrückung bestimmter Organismen (Nitritoxidierer). Aus diesen Gründen ist der Anammox-Prozess zwar vielversprechend, die praktische Implementierung ist jedoch Gegenstand aktiver Forschungs- und Optimierungsarbeiten. Ein weiterer neuer Prozess, die nitrit-abhängige anaerobe Methanoxidation (n-damo), wird großtechnisch noch nicht eingesetzt. Insbesondere in Kombination mit Anammox könnte der n-damo Prozess zur gleichzeitigen Eliminierung von restlichem Methan und von Stickstoff interessant werden.

  • Nitrospira

    2018 - Trends Microbiol., 5: 462-463

    Abstract: 

    In this infographic, the key metabolic functions of Nitrospira and the role that these bacteria play in nitrification and other processes in the environment is shown. Nitrospira plays pivotal roles in nitrification as an aerobic chemolithoautotrophic nitrite-oxidizing bacterium. These bacteria often occur in close association with ammonia-oxidizing bacteria or archaea that convert ammonia to nitrite, which is further oxidized to nitrate by Nitrospira. However, in 'reciprocal feeding' interactions, Nitrospira can also provide ammonia oxidizers with ammonia released from urea or cyanate, which is further nitrified as described above. Recently discovered Nitrospira members even catalyze both nitrification steps alone and are therefore called complete ammonia oxidizers or 'comammox' organisms. Some strains of Nitrospira utilize alternative substrates, such as H and formate, using oxygen or nitrate as terminal electron acceptor, and can exploit these energy sources concurrently with aerobic nitrite oxidation. This metabolic versatility enables Nitrospira to colonize a broad range of habitats and to sustain shifts in environmental conditions such as changing oxygen concentrations.