Publications in peer reviewed journals

33 Publications found
  • Conductive consortia

    2015 - Nature News & Views, 526: 513-514
    anaerobic methane oxidation


    Physiological analyses, electron microscopy and single-cell chemical imaging suggest that direct electron transfer occurs between the members of methane-oxidizing microbial consortia.

  • Genomic Analysis of Xylose Metabolism in Members of the Deinoccocus-Thermus Phylum from Thermophilic Biomass-Deconstructing Bacterial Consortia

    Wu YW, Joshua C, Eichorst SA, Gladden JM, Simmons BA, Singer SW
    2015 - BioEnergy Research, 8: 1031-1038
  • Intestinal microbiota signatures associated with inflammation history in mice experiencing recurring colitis

    Berry D, Kuzyk O, Rauch I, Heider S, Schwab C, Hainzl E, Decker T, Müller M, Strobl B, Schleper C, Urich T, Wagner M, Kenner L, Loy A
    2015 - Front Microbiol, 6: 1408


    Acute colitis causes alterations in the intestinal microbiota, but the microbiota is thought to recover after such events. Extreme microbiota alterations are characteristic of human chronic inflammatory bowel diseases, although alterations reported in different studies are divergent and sometimes even contradictory. To better understand the impact of periodic disturbances on the intestinal microbiota and its compositional difference between acute and relapsing colitis, we investigated the beginnings of recurrent inflammation using the dextran sodium sulfate (DSS) mouse model of chemically induced colitis. Using bacterial 16S rRNA gene-targeted pyrosequencing as well as quantitative fluorescence in situ hybridization, we profiled the intestinal and stool microbiota of mice over the course of three rounds of DSS-induced colitis and recovery. We found that characteristic inflammation-associated microbiota could be detected in recovery-phase mice. Successive inflammation episodes further drove the microbiota into an increasingly altered composition post-inflammation, and signatures of colitis history were detectable in the microbiota more sensitively than by pathology analysis. Bacterial indicators of murine colitis history were identified in intestinal and stool samples, with a high degree of consistency between both sample types. Stool may therefore be a promising non-invasive source of bacterial biomarkers that are highly sensitive to inflammation state and history.

  • Complete nitrification by Nitrospira bacteria

    Daims H, Lebedeva EV, Pjevac P, Han P, Herbold C, Albertsen M, Jehmlich N, Palatinszky M, Vierheilig J, Bulaev A, Kirkegaard RH, Bergen MV, Rattei T, Bendinger B, Nielsen PH, Wagner M
    2015 - Nature, 528: 504-509


    Nitrification, the oxidation of ammonia via nitrite to nitrate, has always been considered to be a two-step process catalysed by chemolithoautotrophic microorganisms oxidizing either ammonia or nitrite. No known nitrifier carries out both steps, although complete nitrification should be energetically advantageous. This functional separation has puzzled microbiologists for a century. Here we report on the discovery and cultivation of a completely nitrifying bacterium from the genus Nitrospira, a globally distributed group of nitrite oxidizers. The genome of this chemolithoautotrophic organism encodes the pathways both for ammonia and nitrite oxidation, which are concomitantly activated during growth by ammonia oxidation to nitrate. Genes affiliated with the phylogenetically distinct ammonia monooxygenase and hydroxylamine dehydrogenase genes of Nitrospira are present in many environments and were retrieved on Nitrospira-contigs in new metagenomes from engineered systems. These findings fundamentally change our picture of nitrification and point to completely nitrifying Nitrospira as key components of nitrogen-cycling microbial communities.

  • Prediction of microbial phenotypes based on comparative genomics

    Feldbauer R, Schulz F, Horn M, Rattei T
    2015 - BMC Bioinformatics, 16: S1


    The accessibility of almost complete genome sequences of uncultivable microbial species from metagenomes necessitates computational methods predicting microbial phenotypes solely based on genomic data. Here we investigate how comparative genomics can be utilized for the prediction of microbial phenotypes. The PICA framework facilitates application and comparison of different machine learning techniques for phenotypic trait prediction. We have improved and extended PICA's support vector machine plug-in and suggest its applicability to large-scale genome databases and incomplete genome sequences

  • Intestinal epithelial cell tyrosine kinase 2 transduces interleukin-22 signals to protect from acute colitis

    Hainzl E, Stockinger S, Rauch I, Heider S, Berry D, Lassnig C, Schwab C, Rosebrock F, Milinovich G, Schlederer M, Wagner M, Schleper C, Loy A, Urich T, Kenner L, Han X, Decker T, Strobl B, Müller M
    2015 - J Immunol., 195: 5011-5024


    In the intestinal tract, IL-22 activates signal transducer and activator of transcription 3 (STAT3) to promote intestinal epithelial cell (IEC) homeostasis and tissue healing. The mechanism has remained obscure but we demonstrate that IL-22 acts via tyrosine kinase 2 (Tyk2), a member of the Janus kinase (Jak) family. Using a mouse model for colitis, we show that Tyk2 deficiency is associated with an altered composition of the gut microbiota and exacerbates inflammatory bowel disease (IBD). Colitic Tyk2-/- mice have less phosphorylated STAT3 (pY-STAT3) in colon tissue and their IECs proliferate less efficiently. Tyk2-deficient primary IECs show reduced pY-STAT3 in response to IL-22 stimulation and expression of IL-22-STAT3 target genes is reduced in IECs from healthy and colitic Tyk2-/- mice. Experiments with conditional Tyk2-/- mice reveal that IEC-specific depletion of Tyk2 aggravates colitis. Disease symptoms can be alleviated by administering high doses of recombinant IL-22-Fc, indicating that Tyk2 deficiency can be rescued via the IL-22 receptor complex. The pivotal function of Tyk2 in IL-22-dependent colitis was confirmed in Citrobacter rodentium-induced disease. Thus, Tyk2 protects against acute colitis in part by amplifying inflammation-induced epithelial IL-22 signaling to STAT3. 

  • Following the footsteps of chlamydial gene regulation

    Domman D, Horn M
    2015 - Mol. Biol. Evol., 32: 3035-3046


    Regulation of gene expression ensures an organism responds to stimuli and undergoes proper development. Although the regulatory networks in bacteria have been investigated in model microorganisms, nearly nothing is known about the evolution and plasticity of these networks in obligate, intracellular bacteria. The phylum Chlamydiae contains a vast array of host-associated microbes, including several human pathogens. The Chlamydiae are unique among obligate, intracellular bacteria as they undergo a complex biphasic developmental cycle in which large swaths of genes are temporally regulated. Coupled with the low number of transcription factors, these organisms offer a model to study the evolution of regulatory networks in intracellular organisms. We provide the first comprehensive analysis exploring the diversity and evolution of regulatory networks across the phylum. We utilized a comparative genomics approach to construct predicted coregulatory networks, which unveiled genus- and family-specific regulatory motifs and architectures, most notably those of virulence-associated genes. Surprisingly, our analysis suggests that few regulatory components are conserved across the phylum, and those that are conserved are involved in the exploitation of the intracellular niche. Our study thus lends insight into a component of chlamydial evolution that has otherwise remained largely unexplored.

  • Abundant toxin-related genes in the genomes of beneficial symbionts from deep-sea hydrothermal vent mussels.

    Sayavedra L, Kleiner M, Ponnudurai R, Wetzel S, Pelletier E, Barbe V, Satoh N, Shoguchi E, Fink D, Breusing C, Reusch TB, Rosenstiel P, Schilhabel MB, Becher D, Schweder T, Markert S, Dubilier N, Petersen JM
    2015 - Elife, e07966


    Bathymodiolus mussels live in symbiosis with intracellular sulfur-oxidizing (SOX) bacteria that provide them with nutrition. We sequenced the SOX symbiont genomes from two Bathymodiolus species. Comparison of these symbiont genomes with those of their closest relatives revealed that the symbionts have undergone genome rearrangements, and up to 35% of their genes may have been acquired by horizontal gene transfer. Many of the genes specific to the symbionts were homologs of virulence genes. We discovered an abundant and diverse array of genes similar to insecticidal toxins of nematode and aphid symbionts, and toxins of pathogens such as Yersinia and Vibrio. Transcriptomics and proteomics revealed that the SOX symbionts express the toxin-related genes (TRGs) in their hosts. We hypothesize that the symbionts use these TRGs in beneficial interactions with their host, including protection against parasites. This would explain why a mutualistic symbiont would contain such a remarkable 'arsenal' of TRGs.

  • Advancements in the application of NanoSIMS and Raman microspectroscopy to investigate the activity of microbial cells in soils

    Eichorst SA, Strasser F, Woyke T, Schintlmeister A, Wagner M, Woebken D
    2015 - FEMS Microbiology Ecology - *Editor's Choice Article*, in press


    The combined approach of incubating environmental samples with stable isotope-labeled substrates followed by single-cell analyses through high-resolution secondary ion mass spectrometry (NanoSIMS) or Raman microspectroscopy provides insights into the in situ function of microorganisms. This approach has found limited application in soils presumably due to the dispersal of microbial cells in a large background of particles. We developed a pipeline for the efficient preparation of cell extracts from soils for subsequent single-cell methods by combining cell detachment with separation of cells and soil particles followed by cell concentration. The procedure was evaluated by examining its influence on cell recoveries and microbial community composition across two soils. This approach generated a cell fraction with considerably reduced soil particle load and of sufficient small size to allow single-cell analysis by NanoSIMS, as shown when detecting active N2-fixing and cellulose-responsive microorganisms via 15N2 and 13C-UL-cellulose incubations, respectively. The same procedure was also applicable for Raman microspectroscopic analyses of soil microorganisms, assessed via microcosm incubations with a 13C-labeled carbon source and deuterium oxide (D2O, a general activity marker). The described sample preparation procedure enables single-cell analysis of soil microorganisms using NanoSIMS and Raman microspectroscopy, but should also facilitate single-cell sorting and sequencing.

  • Expanded metabolic versatility of ubiquitous nitrite-oxidizing bacteria from the genus Nitrospira

    Koch H, Lücker S, Albertsen M, Kitzinger K, Herbold C, Spieck E, Nielsen PH, Wagner M, Daims H
    2015 - Proc Natl Acad Sci U S A, 112: 11371-11376


    Nitrospira are a diverse group of nitrite-oxidizing bacteria and among the environmentally most widespread nitrifiers. Despite this, they remain scarcely studied and mostly uncultured. Based on genomic and experimental data from Nitrospira moscoviensis representing the ubiquitous Nitrospira lineage II, we identified ecophysiological traits that contribute to the ecological success of Nitrospira. Unexpectedly, N. moscoviensis possesses genes coding for a urease and cleaves urea to ammonia and CO2. Ureolysis was not observed yet in nitrite oxidizers and enables N. moscoviensis to supply ammonia oxidizers lacking urease with ammonia from urea, which is fully nitrified by this consortium through reciprocal feeding. The presence of highly similar urease genes in Nitrospira lenta from activated sludge, in metagenomes from soils and freshwater habitats, and of other ureases in marine nitrite oxidizers, suggests a wide distribution of this extended interaction between ammonia and nitrite oxidizers, which enables nitrite-oxidizing bacteria to indirectly utilize urea as a source of energy. A soluble formate dehydrogenase lends additional ecophysiological flexibility and allows N. moscoviensis to utilize formate, with or without concomitant nitrite oxidation, using oxygen, nitrate, or both compounds as terminal electron acceptors. Compared to Nitrospira defluvii from lineage I, N. moscoviensis shares the Nitrospira core metabolism but shows substantial genomic dissimilarity including genes for adaptations to elevated oxygen concentrations. Reciprocal feeding and metabolic versatility, including the participation in different nitrogen cycling processes, likely are key factors for the niche partitioning, the ubiquity, and the high diversity of Nitrospira in natural and engineered ecosystems.

  • Marine amoebae with cytoplasmic and perinuclear symbionts deeply branching in the Gammaproteobacteria

    Schulz F, Tyml T, Pizzetti I, Dyková I, Fazi S, Kostka M, Horn M
    2015 - Sci Rep, 5: 13381


    Amoebae play an important ecological role as predators in microbial communities. They also serve as niche for bacterial replication, harbor endosymbiotic bacteria and have contributed to the evolution of major human pathogens. Despite their high diversity, marine amoebae and their association with bacteria are poorly understood. Here we describe the isolation and characterization of two novel marine amoebae together with their bacterial endosymbionts, tentatively named 'Candidatus Occultobacter vannellae' and 'Candidatus Nucleophilum amoebae'. While one amoeba strain is related to Vannella, a genus common in marine habitats, the other represents a novel lineage in the Amoebozoa. The endosymbionts showed only low similarity to known bacteria (85-88% 16S rRNA sequence similarity) but together with other uncultured marine bacteria form a sister clade to the Coxiellaceae. Using fluorescence in situ hybridization and transmission electron microscopy, identity and intracellular location of both symbionts were confirmed; one was replicating in host-derived vacuoles, whereas the other was located in the perinuclear space of its amoeba host. This study sheds for the first time light on a so far neglected group of protists and their bacterial symbionts. The newly isolated strains represent easily maintainable model systems and pave the way for further studies on marine associations between amoebae and bacterial symbionts.

  • Endosymbionts escape dead hydrothermal vent tubeworms to enrich the free-living population.

    Klose J, Polz MF, Wagner M, Schimak MP, Gollner S, Bright M
    2015 - Proc. Natl. Acad. Sci. U.S.A., 36: 11300-11305


    Theory predicts that horizontal acquisition of symbionts by plants and animals must be coupled to release and limited dispersal of symbionts for intergenerational persistence of mutualisms. For deep-sea hydrothermal vent tubeworms (Vestimentifera, Siboglinidae), it has been demonstrated that a few symbiotic bacteria infect aposymbiotic host larvae and grow in a newly formed organ, the trophosome. However, whether viable symbionts can be released to augment environmental populations has been doubtful, because (i) the adult worms lack obvious openings and (ii) the vast majority of symbionts has been regarded as terminally differentiated. Here we show experimentally that symbionts rapidly escape their hosts upon death and recruit to surfaces where they proliferate. Estimating symbiont release from our experiments taken together with well-known tubeworm density ranges, we suggest a few million to 1.5 billion symbionts seeding the environment upon death of a tubeworm clump. In situ observations show that such clumps have rapid turnover, suggesting that release of large numbers of symbionts may ensure effective dispersal to new sites followed by active larval colonization. Moreover, release of symbionts might enable adaptations that evolve within host individuals to spread within host populations and possibly to new environments.

  • Draft genome sequence of 'Candidatus Hepatoplasma crinochetorum Ps' – a bacterial symbiont in the hepatopancreas of the terrestrial isopod Porcellio scaber

    Collingro A, Kostanjšek R, Toenshoff ER, Schulz F, Schuster L, Domann D, Horn M
    2015 - Genome Announc, 3: e00674-15


    'Candidatus Hepatoplasma crinochetorum Ps' is an extracellular symbiont residing in the hepatopancreas of the terrestrial isopod Porcellio scaber. Its genome is highly similar to that of the close relative 'Cand. Hepatoplasma crinochetorum Av' from Armadillidium vulgare. However, instead of a CRISPR/Cas system it encodes a type I restriction modification system.

  • A flexible and economical barcoding approach for highly multiplexed amplicon sequencing of diverse target genes.

    Herbold CW, Pelikan C, Kuzyk O, Hausmann B, Angel R, Berry D, Loy A
    2015 - Front Microbiol, 6: 731


    High throughput sequencing of phylogenetic and functional gene amplicons provides tremendous insight into the structure and functional potential of complex microbial communities. Here, we introduce a highly adaptable and economical PCR approach to barcoding and pooling libraries of numerous target genes. In this approach, we replace gene- and sequencing platform-specific fusion primers with general, interchangeable barcoding primers, enabling nearly limitless customized barcode-primer combinations. Compared to barcoding with long fusion primers, our multiple-target gene approach is more economical because it overall requires lower number of primers and is based on short primers with generally lower synthesis and purification costs. To highlight our approach, we pooled over 900 different small-subunit rRNA and functional gene amplicon libraries obtained from various environmental or host-associated microbial community samples into a single, paired-end Illumina MiSeq run. Although the amplicon regions ranged in size from approximately 290 to 720 bp, we found no significant systematic sequencing bias related to amplicon length or gene target. Our results indicate that this flexible multiplexing approach produces large, diverse, and high quality sets of amplicon sequence data for modern studies in microbial ecology.

  • Cyanate as an energy source for nitrifiers.

    Palatinszky M, Herbold C, Jehmlich N, Pogoda M, Han P, von Bergen M, Lagkouvardos I, Karst SM, Galushko A, Koch H, Berry D, Daims H, Wagner M
    2015 - Nature, 524: 105-108


    Ammonia- and nitrite-oxidizing microorganisms are collectively responsible for the aerobic oxidation of ammonia via nitrite to nitrate and have essential roles in the global biogeochemical nitrogen cycle. The physiology of nitrifiers has been intensively studied, and urea and ammonia are the only recognized energy sources that promote the aerobic growth of ammonia-oxidizing bacteria and archaea. Here we report the aerobic growth of a pure culture of the ammonia-oxidizing thaumarchaeote Nitrososphaera gargensis using cyanate as the sole source of energy and reductant; to our knowledge, the first organism known to do so. Cyanate, a potentially important source of reduced nitrogen in aquatic and terrestrial ecosystems, is converted to ammonium and carbon dioxide in Nitrososphaera gargensis by a cyanase enzyme that is induced upon addition of this compound. Within the cyanase gene family, this cyanase is a member of a distinct clade also containing cyanases of nitrite-oxidizing bacteria of the genus Nitrospira. We demonstrate by co-culture experiments that these nitrite oxidizers supply cyanase-lacking ammonia oxidizers with ammonium from cyanate, which is fully nitrified by this microbial consortium through reciprocal feeding. By screening a comprehensive set of more than 3,000 publically available metagenomes from environmental samples, we reveal that cyanase-encoding genes clustering with the cyanases of these nitrifiers are widespread in the environment. Our results demonstrate an unexpected metabolic versatility of nitrifying microorganisms, and suggest a previously unrecognized importance of cyanate in cycling of nitrogen compounds in the environment.

  • Methanogenic archaea and sulfate reducing bacteria co-cultured on acetate: teamwork or coexistence?

    Ozuolmez D, Na H, Lever MA, Kjeldsen KU, Jørgensen BB, Plugge CM
    2015 - Front Microbiol, 6: 492


    Acetate is a major product of fermentation processes and an important substrate for sulfate reducing bacteria and methanogenic archaea. Most studies on acetate catabolism by sulfate reducers and methanogens have used pure cultures. Less is known about acetate conversion by mixed pure cultures and the interactions between both groups. We tested interspecies hydrogen transfer and coexistence between marine methanogens and sulfate reducers using mixed pure cultures of two types of microorganisms. First, Desulfovibrio vulgaris subsp. vulgaris (DSM 1744), a hydrogenotrophic sulfate reducer, was cocultured together with the obligate aceticlastic methanogen Methanosaeta concilii using acetate as carbon and energy source. Next, Methanococcus maripaludis S2, an obligate H2- and formate-utilizing methanogen, was used as a partner organism to M. concilii in the presence of acetate. Finally, we performed a coexistence experiment between M. concilii and an acetotrophic sulfate reducer Desulfobacter latus AcSR2. Our results showed that D. vulgaris was able to reduce sulfate and grow from hydrogen leaked by M. concilii. In the other coculture, M. maripaludis was sustained by hydrogen leaked by M. concilii as revealed by qPCR. The growth of the two aceticlastic microbes indicated co-existence rather than competition. Altogether, our results indicate that H2 leaking from M. concilii could be used by efficient H2-scavengers. This metabolic trait, revealed from coculture studies, brings new insight to the metabolic flexibility of methanogens and sulfate reducers residing in marine environments in response to changing environmental conditions and community compositions. Using dedicated physiological studies we were able to unravel the occurrence of less obvious interactions between marine methanogens and sulfate-reducing bacteria.

  • Uncultured Desulfobacteraceae and Crenarchaeotal group C3 incorporate 13 C-acetate in coastal marine sediment

    Na H, Lever MA, Kjeldsen KU, Schulz F, Jørgensen BB
    2015 - Environ Microbiol Rep, 7: 614-22


    Stable isotope probing (SIP) of deoxyribonucleic acid (DNA) was used to identify microbes incorporating 13 C-labeled acetate in sulfate-reducing sediment from Aarhus Bay, Denmark. Sediment was incubated in medium containing 10 mM sulfate and different 13 C-acetate (10, 1, 0.1 mM) concentrations. The resultant changes in microbial community composition were monitored in total and SIP-fractionated DNA during long-term incubations. Chemical analyses demonstrated metabolic activity in all sediment slurries, with sulfate-reducing activity largely determined by initial acetate concentrations. Sequencing of 16S rRNA gene PCR amplicons showed that the incubations shifted the bacterial but not the archaeal community composition. After 3 months of incubation, only sediment slurries incubated with 10 mM 13 C-acetate showed detectable 13 C-DNA labeling. Based on 16S rRNA and dsrB gene PCR amplicon sequencing, the 13 C-labeled DNA pool was dominated by a single type of sulfate reducer representing a novel genus in the family Desulfobacteraceae. In addition, members of the uncultivated Crenarchaeotal group C3 were enriched in the 13 C-labeled DNA. Our results were reproducible across biological replicate experiments and provide new information about the identities of uncultured acetate-consuming bacteria and archaea in marine sediments.

  • Single-cell genomics of a rare environmental alphaproteobacterium provides unique insights into Rickettsiaceae evolution

    Martijn J, Schulz F, Zaremba-Niedzwiedzka K, Viklund J, Stepanauskas R, Andersson SG, Horn M, Guy L, Ettema TJ
    2015 - ISME J, 9: 2373-85


    The bacterial family Rickettsiaceae includes a group of well-known etiological agents of many human and vertebrate diseases, including epidemic typhus-causing pathogen Rickettsia prowazekii. Owing to their medical relevance, rickettsiae have attracted a great deal of attention and their host-pathogen interactions have been thoroughly investigated. All known members display obligate intracellular lifestyles, and the best-studied genera, Rickettsia and Orientia, include species that are hosted by terrestrial arthropods. Their obligate intracellular lifestyle and host adaptation is reflected in the small size of their genomes, a general feature shared with all other families of the Rickettsiales. Yet, despite that the Rickettsiaceae and other Rickettsiales families have been extensively studied for decades, many details of the origin and evolution of their obligate host-association remain elusive. Here we report the discovery and single-cell sequencing of 'Candidatus Arcanobacter lacustris', a rare environmental alphaproteobacterium that was sampled from Damariscotta Lake that represents a deeply rooting sister lineage of the Rickettsiaceae. Intriguingly, phylogenomic and comparative analysis of the partial 'Candidatus Arcanobacter lacustris' genome revealed the presence chemotaxis genes and vertically inherited flagellar genes, a novelty in sequenced Rickettsiaceae, as well as several host-associated features. This finding suggests that the ancestor of the Rickettsiaceae might have had a facultative intracellular lifestyle. Our study underlines the efficacy of single-cell genomics for studying microbial diversity and evolution in general, and for rare microbial cells in particular.

  • Plastid establishment did not require a chlamydial partner

    Domman D, Horn M, Embley TM, Williams TA
    2015 - Nat Commun, 6: 6421


    Primary plastids descend from the cyanobacterial endosymbiont of an ancient eukaryotic host, but the initial selective drivers that stabilized the association between these two cells are still unclear. One hypothesis that has achieved recent prominence suggests that the first role of the cyanobiont was in energy provision for a host cell whose reserves were being depleted by an intracellular chlamydial pathogen. A pivotal claim is that it was chlamydial proteins themselves that converted otherwise unusable cyanobacterial metabolites into host energy stores. We test this hypothesis by investigating the origins of the key enzymes using sophisticated phylogenetics. Here we show a mosaic origin for the relevant pathway combining genes with host, cyanobacterial or bacterial ancestry, but we detect no strong case for Chlamydiae to host transfer under the best-fitting models. Our conclusion is that there is no compelling evidence from gene trees that Chlamydiae played any role in establishing the primary plastidendosymbiosis.

  • Dimeric chlorite dismutase from the nitrogen-fixing cyanobacterium Cyanothece sp. PCC7425

    Schaffner I, Hofbauer S, Krutzler M, Pirker KF, Bellei M, Stadlmayr G, Mlynek G, Djinovic-Carugo K, Battistuzzi G, Furtmüller PG, Daims H, Obinger C
    2015 - Mol Microbiol, 96: 1053-1068


    It is demonstrated that cyanobacteria (both azotrophic and non-azotrophic) contain heme b oxidoreductases that can convert chlorite to chloride and molecular oxygen (incorrectly denominated chlorite 'dismutase', Cld). Beside the water-splitting manganese complex of photosystem II, this metalloenzyme is the second known enzyme that catalyses the formation of a covalent oxygen-oxygen bond. All cyanobacterial Clds have a truncated N-terminus and are dimeric (i.e. clade 2) proteins. As model protein, Cld from Cyanothece sp. PCC7425 (CCld) was recombinantly produced in Escherichia coli and shown to efficiently degrade chlorite with an activity optimum at pH 5.0 [kcat 1144 ± 23.8 s-1 , KM 162 ± 10.0 μM, catalytic efficiency (7.1 ± 0.6) × 106  M-1  s-1 ]. The resting ferric high-spin axially symmetric heme enzyme has a standard reduction potential of the Fe(III)/Fe(II) couple of -126 ± 1.9 mV at pH 7.0. Cyanide mediates the formation of a low-spin complex with kon  = (1.6 ± 0.1) × 105  M-1  s-1 and koff  = 1.4 ± 2.9 s-1 (KD  ∼ 8.6 μM). Both, thermal and chemical unfolding follows a non-two-state unfolding pathway with the first transition being related to the release of the prosthetic group. The obtained data are discussed with respect to known structure-function relationships of Clds. We ask for the physiological substrate and putative function of these O2 -producing proteins in (nitrogen-fixing) cyanobacteria.

  • Intranuclear bacteria: inside the cellular control center of eukaryotes

    Schulz F, Horn M
    2015 - Trends Cell Biol, 25: 339-346


    Intracellular bacteria including major pathogens live in the cytoplasm or in cytoplasmic vacuoles within their host cell. However, some can invade more unusual intracellular niches such as the eukaryotic nucleus. Phylogenetically diverse intranuclear bacteria have been discovered in various protist, arthropod, marine invertebrate, and mammalian hosts. Although targeting the same cellular compartment, they have apparently developed fundamentally-different infection strategies. The nucleus provides a rich pool of nutrients and protection against host cytoplasmic defense mechanisms; intranuclear bacteria can directly manipulate the host by interfering with nuclear processes. The impact on their host cells ranges from stable associations with a neutral or beneficial effect on host fitness to rapid host lysis. The analysis of the intranuclear lifestyle will extend our current framework for understanding host-pathogen interactions.

  • Structure and heme-binding properties of HemQ (chlorite dismutase-like protein) from Listeria monocytogenes

    Hofbauer S, Hagmüller A, Schaffner I, Mlynek G, Krutzler M, Stadlmayr G, Pirker KF, Obinger C, Daims H, Djinovic-Carugo K, Furtmüller PG
    2015 - Arch Biochem Biophys, 574: 36-48


    Chlorite dismutase-like proteins are structurally closely related to functional chlorite dismutases which are heme b-dependent oxidoreductases capable of reducing chlorite to chloride with simultaneous production of dioxygen. Chlorite dismutase-like proteins are incapable of performing this reaction and their biological role is still under discussion. Recently, members of this large protein family were shown to be involved in heme biosynthesis in Gram-positive bacteria, and thus the protein was renamed HemQ in these organisms. In the present work the structural and heme binding properties of the chlorite dismutase-like protein from the Gram-positive pathogen Listeria monocytogenes (LmCld) were analyzed in order to evaluate its potential role as a regulatory heme sensing protein. The homopentameric crystal structure (2.0Å) shows high similarity to chlorite-degrading chlorite dismutases with an important difference in the structure of the putative substrate and heme entrance channel. In solution LmCld is a stable hexamer able to bind the low-spin ligand cyanide. Heme binding is reversible with KD-values determined to be 7.2μM (circular dichroism spectroscopy) and 16.8μM (isothermal titration calorimetry) at pH 7.0. Both acidic and alkaline conditions promote heme release. Presented biochemical and structural data reveal that the chlorite dismutase-like protein from L. monocytogenes could act as a potential regulatory heme sensing and storage protein within heme biosynthesis.

  • Inhibitory properties of C2 - C10 1-alkynes on ammonia oxidation in two Nitrososphaera species

    Taylor A, Taylor K, Tennigkeit B, Palatinszky M, Stieglmeier M, Myrold D, Schleper C, Wagner M, Bottomley P
    2015 - Appl Environ Microbiol, 81: 1942-8


    A previous study showed that ammonia oxidation by the Thaumarchaeota Nitrosopumilus maritimus (group 1.1a) was resistant to concentrations of the C8 1-alkyne, octyne, which completely inhibits activity by ammonia-oxidizing bacteria. In this study, the inhibitory effects of octyne and other C2 to C10 1-alkynes were evaluated on the nitrite production activity of two pure culture isolates from Thaumarchaeota group 1.1b, Nitrososphaera viennensis strain EN76 and Nitrososphaera gargensis. Both N. viennensis and N. gargensis were insensitive to concentrations of octyne that cause complete and irreversible inactivation of nitrite production by ammonia-oxidizing bacteria. However, octyne concentrations (≥20 μM) that did not inhibit N. maritimus partially inhibited nitrite production in N. viennensis and N. gargensis in a manner that did not show the characteristics of irreversible inactivation. In contrast to previous studies with an ammonia-oxidizing bacterium, Nitrosomonas europaea, octyne inhibition of N. viennensis was: (i) fully and immediately reversible, (ii) not competitive with NH4 (+), and (iii) without effect on the competitive interaction between NH4 (+) and acetylene. Both N. viennensis and N. gargensis demonstrated the same overall trend in regard to 1-alkyne inhibition as previously observed for N. maritimus, being highly sensitive to ≤C5 alkynes and more resistant to longer-chain length alkynes. Reproducible differences were observed among N. maritimus, N. viennensis, and N. gargensis in regard to the extent of their resistance/sensitivity to C6 and C7 1-alkynes, which may indicate differences in the ammonia monooxygenase binding and catalytic site(s) among the Thaumarchaeota.

  • Tracking heavy water (D2O) incorporation for identifying and sorting active microbial cells

    Berry D, Mader E, Lee TK, Woebken D, Wang Y, Zhu D, Palatinszky M, Schintlmeister A, Schmid MC, Hanson BT, Shterzer N, Mizrahi I, Rauch I, Decker T, Bocklitz T, Popp J, Gibson CM, Fowler PW, Huang WE, Wagner M
    2015 - Proc Natl Acad Sci USA, 112: E194-203


    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 nanoscale-resolution 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 Raman-based cell sortingof 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 sortingof microbial cells with defined functional properties for single-cell genomics.

  • Comparison of oxidation kinetics of nitrite-oxidizing bacteria: nitrite availability as a key factor in niche differentiation

    Nowka B, Daims H, Spieck E
    2015 - Appl Environ Microbiol, 81: 745-53


    Nitrification has an immense impact on nitrogen cycling in natural ecosystems and in wastewater treatment plants. Mathematical models function as tools to capture the complexity of these biological systems, but kinetic parameters especially of nitrite-oxidizing bacteria (NOB) are lacking because of a limited number of pure cultures until recently. In this study, we compared the nitrite oxidation kinetics of six pure cultures and one enrichment culture representing three genera of NOB (Nitrobacter, Nitrospira, Nitrotoga). With half-saturation constants (Km) between 9 and 27 μM nitrite, Nitrospira bacteria are adapted to live under significant substrate limitation. Nitrobacter showed a wide range of lower substrate affinities, with Km values between 49 and 544 μM nitrite. However, the advantage of Nitrobacter emerged under excess nitrite supply, sustaining high maximum specific activities (Vmax) of 64 to 164 μmol nitrite/mg protein/h, contrary to the lower activities of Nitrospira of 18 to 48 μmol nitrite/mg protein/h. The Vmax (26 μmol nitrite/mg protein/h) and Km (58 μM nitrite) of "Candidatus Nitrotoga arctica" measured at a low temperature of 17°C suggest that Nitrotoga can advantageously compete with other NOB, especially in cold habitats. The kinetic parameters determined represent improved basis values for nitrifying models and will support predictions of community structure and nitrification rates in natural and engineered ecosystems.

  • Phylogenetic and environmental diversity of DsrAB-type dissimilatory (bi)sulfite reductases

    Müller AL, Kjeldsen KU, Rattei T, Pester M, Loy A
    2015 - ISME J, 9: 1152-1165


    The energy metabolism of essential microbial guilds in the biogeochemical sulfur cycle is based on a DsrAB-type dissimilatory (bi)sulfite reductase that either catalyzes the reduction of sulfite to sulfide during anaerobic respiration of sulfate, sulfite and organosulfonates, or acts in reverse during sulfur oxidation. Common use of dsrAB as a functional marker showed that dsrAB richness in many environments is dominated by novel sequence variants and collectively represents an extensive, largely uncharted sequence assemblage. Here, we established a comprehensive, manually curated dsrAB/DsrAB database and used it to categorize the known dsrAB diversity, reanalyze the evolutionary history of dsrAB and evaluate the coverage of published dsrAB-targeted primers. Based on a DsrAB consensus phylogeny, we introduce an operational classification system for environmentaldsrAB sequences that integrates established taxonomic groups with operational taxonomic units (OTUs) at multiple phylogenetic levels, ranging from DsrAB enzyme families that reflect reductive or oxidative DsrAB types of bacterial or archaeal origin, superclusters, uncultured family-level lineages to species-level OTUs. Environmental dsrAB sequences constituted at least 13 stable family-level lineages without any cultivated representatives, suggesting that major taxa of sulfite/sulfate-reducing microorganisms have not yet been identified. Three of these uncultured lineages occur mainly in marine environments, while specific habitat preferences are not evident for members of the other 10 uncultured lineages. In summary, our publically available dsrAB/DsrAB database, the phylogenetic framework, the multilevel classification system and a set of recommended primers provide a necessary foundation for large-scale dsrAB ecology studies with next-generation sequencing methods.

  • Revisiting N₂ fixation in Guerrero Negro intertidal microbial mats with a functional single-cell approach

    Woebken D, Burow L, Behnam F, Mayali X, Schintlmeister A, Fleming E, Prufert-Bebout L, Singer S, López Cortés A, Hoehler T, Pett-Ridge J, Spormann A, Wagner M, Weber P, Bebout B
    2015 - ISME J, 9: 485-96


    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 inintertidal 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.

  • Conserved features and major differences in the outer membrane protein composition of chlamydiae

    Aistleitner K, Anrather D, Schott T, Klose J, Bright M, Ammerer G, Horn M
    2015 - Environ Microbiol, 7: 1397-413


    Chlamydiae are a highly successful group of obligate intracellular bacteria infecting a variety of eukaryotic hosts. Outer membrane proteins involved in attachment to and uptake into host cells, and cross-linking of these proteins via disulfide bonds are key features of the biphasic chlamydial developmental cycle. In this study, we used a consensus approach to predict outer membrane proteins in the genomes of members of three chlamydial families. By analysing outer membrane protein fractions of purified chlamydiae with highly sensitive mass spectrometry, we show that theprotein composition differs strongly between these organisms. Large numbers of major outer membrane protein-like proteins are present at high abundance in the outer membrane of Simkania negevensis and Waddlia chondrophila, whereas yet uncharacterized putative porins dominate in Parachlamydia acanthamoebae. Simkania represents the first case of a chlamydia completely lacking stabilizing cysteine-rich proteins in its outermembrane. In agreement with this, and in contrast to Parachlamydia and Waddlia, the cellular integrity of Simkania is not impaired by conditions that reduce disulfide bonds of these proteins. The observed differences in the protein composition of the outer membrane among members of divergent chlamydial families suggest different stabilities of these organisms in the environment, probably due to adaption to different niches or transmission routes.

  • Nitrotoga-like bacteria are previously unrecognized key nitrite oxidizers in full-scale wastewater treatment plants

    Lücker S, Schwarz J, Gruber-Dorninger C, Spieck E, Wagner M, Daims H
    2015 - ISME J, 9: 708-720


    Numerous past studies have shown members of the genus Nitrospira to be the predominant nitrite-oxidizing bacteria (NOB) in nitrifying wastewatertreatment plants (WWTPs). Only recently, the novel NOB 'Candidatus Nitrotoga arctica' was identified in permafrost soil and a close relative was enriched from activated sludge. Still, little is known about diversity, distribution and functional importance of Nitrotoga in natural and engineered ecosystems. Here we developed Nitrotoga 16S rRNA-specific PCR primers and fluorescence in situ hybridization (FISH) probes, which were applied to screen activated sludge samples from 20 full-scale WWTPs. Nitrotoga-like bacteria were detected by PCR in 11 samples and reached abundances detectable by FISH in seven sludges. They coexisted with Nitrospira in most of these WWTPs, but constituted the only detectable NOB in two systems. Quantitative FISH revealed that Nitrotoga accounted for nearly 2% of the total bacterial community in one of these plants, a number comparable to Nitrospira abundances in other WWTPs. Spatial statistics revealed that Nitrotoga coaggregated with ammonia-oxidizing bacteria, strongly supporting a functional role in nitrite oxidation. This activity was confirmed by FISH in combination with microradiography, which revealednitrite-dependent autotrophic carbon fixation by Nitrotoga in situ. Correlation of the presence or absence with WWTP operational parameters indicated low temperatures as a main factor supporting high Nitrotoga abundances, although in incubation experiments these NOB remained active over an unexpected range of temperatures, and also at different ambient nitrite concentrations. In conclusion, this study demonstrates that Nitrotoga can be functionally important nitrite oxidizers in WWTPs and can even represent the only known NOB in engineered systems.

  • Functionally relevant diversity of closely related Nitrospira in activated sludge

    Gruber-Dorninger C, Pester M, Kitzinger K, Savio DF, Loy A, Rattei T, Wagner M, Daims H
    2015 - ISME J, 9: 643-655


    Nitrospira are chemolithoautotrophic nitrite-oxidizing bacteria that catalyze the second step of nitrification in most oxic habitats and are important for excess nitrogen removal from sewage in wastewater treatment plants (WWTPs). To date, little is known about their diversity and ecological niche partitioning within complex communities. In this study, the fine-scale community structure and function of Nitrospira was analyzed in two full-scale WWTPs as model ecosystems. In Nitrospira-specific 16S rRNA clone libraries retrieved from each plant, closely related phylogenetic clusters (16S rRNA identities between clusters ranged from 95.8% to 99.6%) within Nitrospira lineages I and II were found. Newly designed probes for fluorescence in situ hybridization (FISH) allowed the specific detection of several of these clusters, whose coexistence in the WWTPs was shown for prolonged periods of several years. In situ ecophysiological analyses based on FISH, relative abundance and spatial arrangement quantification, as well as microautoradiography revealed functional differences of these Nitrospira clusters regarding the preferred nitrite concentration, the utilization of formate as substrate and the spatial coaggregation with ammonia-oxidizing bacteria as symbiotic partners. Amplicon pyrosequencing of the nxrB gene, which encodes subunit beta of nitrite oxidoreductase of Nitrospira, revealed in one of the WWTPs as many as 121 species-level nxrB operational taxonomic units with highly uneven relative abundances in the amplicon library. These results show a previously unrecognized highdiversity of Nitrospira in engineered systems, which is at least partially linked to niche differentiation and may have important implications for process stability.

  • A nanoscale secondary ion mass spectrometry study of dinoflagellate functional diversity in reef-building corals.

    Pernice M, Dunn SR, Tonk L, Dove S, Domart-Coulon I, Hoppe P, Schintlmeister A, Wagner M, Meibom A
    2015 - Environ. Microbiol., 10: 3570-80


    Nutritional interactions between corals and symbiotic dinoflagellate algae lie at the heart of the structural foundation of coral reefs. Whilst the genetic diversity of Symbiodinium has attracted particular interest because of its contribution to the sensitivity of corals to environmental changes and bleaching (i.e. disruption of coral-dinoflagellate symbiosis), very little is known about the in hospite metabolic capabilities of different Symbiodinium types. Using a combination of stable isotopic labelling and nanoscale secondary ion mass spectrometry (NanoSIMS), we investigated the ability of the intact symbiosis between the reef-building coral Isopora palifera, and Symbiodinium C or D types, to assimilate dissolved inorganic carbon (via photosynthesis) and nitrogen (as ammonium). Our results indicate that Symbiodinium types from two clades naturally associated with I. palifera possess different metabolic capabilities. The Symbiodinium C type fixed and passed significantly more carbon and nitrogen to its coral host than the D type. This study provides further insights into the metabolic plasticity among different Symbiodinium types in hospite and strengthens the evidence that the more temperature-tolerant Symbiodinium D type may be less metabolically beneficial for its coral host under non-stressful conditions.

  • Improved isolation strategies allowed the phenotypic differentiation of two Nitrospira strains from widespread phylogenetic lineages.

    Nowka B, Off S, Daims H, Spieck E
    2015 - FEMS Microbiol Ecol, 91: fiu031


    The second step of nitrification, the oxidation of nitrite to nitrate, is vital for the functioning of the nitrogen cycle, but our understanding of the ecological roles of the involved microorganisms is still limited. The known diversity of Nitrospira, the most widely distributed nitrite-oxidizing bacteria, has increased remarkably by analyses of 16S rRNA and functional gene sequences. However, only few representatives could be brought into laboratory cultures so far. In this study, two Nitrospira from activated sludge were isolated using novel approaches together with established methods. Highly enriched 'Candidatus Nitrospira defluvii' was separated from concomitant heterotrophs by taking advantage of its resistance against ampicillin and acriflavine. Beside this member of lineage I, a novel species of lineage II, named N. lenta, was initially enriched at 10°C and finally purified by using optical tweezers. The tolerance to elevated nitrite levels was much higher in N. defluvii than in the more fastidious N. lenta and was accompanied by pronounced biofilm formation. Phylogenetic classification of 12 additional enrichments indicated that Nitrospira lineage I is common in extreme and moderate ecosystems like lineage II. The new cultures will help to explore physiological and genomic differences affecting niche separation between members of this highly diverse genus.

  • Emendation of the family Chlamydiaceae: Proposal of a single genus, Chlamydia, to include all currently recognized species

    Sachse K, Bavoil PM, Kaltenboeck B, Stephens RS, Kuo CC, Rosselló-Móra R, Horn M
    2015 - Syst Appl Microbiol, 38: 99-103


    The family Chlamydiaceae (order Chlamydiales, phylum Chlamydiae) comprises important, obligate intracellular bacterial pathogens of humans and animals. Subdivision of the family into the two genera Chlamydia and Chlamydophila has been discussed controversially during the past decade. Here, we have revisited the current classification in the light of recent genomic data and in the context of the unique biological properties of these microorganisms. We conclude that neither generally used 16S rRNA sequence identity cut-off values nor parameters based on genomic similarity consistently separate the two genera. Notably, no easily recognizable phenotype such as host preference or tissue tropism is available that would support a subdivision. In addition, the genus Chlamydophila is currently not well accepted and not used by a majority of research groups in the field. Therefore, we propose the classification of all 11 currently recognized Chlamydiaceae species in a single genus, the genus Chlamydia. Finally, we provide emended descriptions of the family Chlamydiaceae, the genus Chlamydia, as well as the species Chlamydia abortus, Chlamydia caviae and Chlamydia felis.

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