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In silico determination of nitrogen metabolism in microbes from extreme conditions using metagenomics

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Abstract

The acid ponds of the Danakil Depression in northern Ethiopia are polyextreme environments that exceed the normal physicochemical limits of pH, salinity, ion content, and temperature. We tested for the occurrence of DNA-based life in this environment using Metagenomic Shotgun DNA sequencing approaches. The obtained sequences were examined by the bioinformatic tools MetaSpades, DIAMOND and MEGAN 6-CE, and we were able to bin more than 90% of the metagenomics contigs of Dallol and Black Water to the Bacteria domain, and to the Proteobacteria phylum. Predictions of gene function based on SEED disclosed the presence of different nutrient cycles in the acid ponds. For this study, we focused on partial or completely sequenced genes involved in nitrogen metabolism. The KEGG nitrogen metabolism pathway mapping results for both acid ponds showed that all the predicted genes are involved directly or indirectly in the assimilation of ammonia and no dissimilation or nitrification process was identified. Furthermore, the deduced nitrogen fixation in the two acid ponds based on SEED classification indicated the presence of different sets of nitrogen fixing (nif) genes for biosynthesis and maturation of nitrogenase. Based on the in silico analysis, the predicted proteins involved in nitrogen fixation, especially the cysteine desulfurase and [4Fe-4S] ferredoxin, from both acid ponds are unique with less than 80% sequence similarity to the next closest protein sequence. Considering the extremity of the environmental conditions of the two acid ponds in the Danakil depression, this metagenomics dataset can add to the study of unique gene functions in nitrogen metabolism that enable thriving biocommunities in hypersaline and highly acidic conditions.

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References

  • Spear JR, Walker JJ, Pace NR (2006) Microbial ecology and energetics in yellowstone hot springs. Yellowstone Sci 14(1):17–24

    Google Scholar 

  • Mayer K et al (2017) Phreatic activity and hydrothermal alteration in the Valley of Desolation, Dominica, Lesser Antilles. Bull Volcanol 79(12):82

    Google Scholar 

  • Crognale S et al (2018) Microbiome profiling in extremely acidic soils affected by hydrothermal fluids: the case of the Solfatara Crater (Campi Flegrei, southern Italy). FEMS Microbiol Ecol 94(12)

  • Illsley-Kemp F et al (2017) Local Earthquake Magnitude Scale and b-Value for the Danakil Region of Northern Afar. Bull Seismol Soc Am pp 107

  • Franzson H, Helgado HM, Oskarsson F (2015) Surface Exploration and First Conceptual Model of the Dallol Geothermal Area, Northern Afar, Ethiopia. In: Proceedings World Geothermal Congress

  • Asrat A (2016) The Danakil depression: an exceptional place where different types of extreme environments coexist. pp 189–196

  • Carrizo D et al (2019) Lipid biomarker and carbon stable isotope survey on the dallol hydrothermal system in Ethiopia. Astrobiology 19(12):1474–1489

    CAS  PubMed  PubMed Central  Google Scholar 

  • Gómez F et al (2019) Ultra-small microorganisms in the polyextreme conditions of the Dallol volcano, Northern Afar, Ethiopia. Sci Rep 9:7907. https://doi.org/10.1038/s41598-019-44440-8

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Damer B (2016) A field trip to the Archaean in search of Darwin’s Warm Little Pond. Life (Basel, Switzerland) 6(2):21

    Google Scholar 

  • Van Kranendonk MJ, Deamer DW, Djokic T (2017) Life springs. Sci Am 317(2):28–35

    PubMed  Google Scholar 

  • Wächtershäuser G (2006) From volcanic origins of chemoautotrophic life to Bacteria, Archaea and Eukarya. Philos Trans R Soc Lond B Biol Sci 361(1474):1787–806; discussion 1806–8

  • Barbieri R, Cavalazzi B (2014) How Do Modern Extreme Hydrothermal Environments Inform the Identification of Martian Habitability? The Case of the El Tatio Geyser Field. Challenges 5:430–443

    Google Scholar 

  • van den Burg B (2003) Extremophiles as a source for novel enzymes. Curr Opin Microbiol 6(3):213–218

    PubMed  Google Scholar 

  • Hamdi M (2007) Microbial Resources and Industrial microbial processes design and behavior. Current Research, Technology and Education Topics in Applied Microbiology and Microbial Biotechnology, A. Mendez-Vilas, Editor. 2007.

  • Kikani B, Shukla R (2010) and S. Biocatalytic potential of thermophilic bacteria and actinomycetes, Singh, pp 1000–1007

    Google Scholar 

  • Börne RA (2013) Exploring anaerobic bacteria for industrial biotechnology: diversity studies, screening and biorefinery applications. Lund University, Sweden

    Google Scholar 

  • Li SJ et al (2014) Microbial communities evolve faster in extreme environments. Sci Rep 4:6205

    CAS  PubMed  PubMed Central  Google Scholar 

  • Schmeisser C, Steele H, Streit WR (2007) Metagenomics, biotechnology with non-culturable microbes. Appl Microbiol Biotechnol 75(5):955–962

    CAS  PubMed  Google Scholar 

  • Su C et al (2012) Culture-independent methods for studying environmental microorganisms: methods, application, and perspective. Appl Microbiol Biotechnol 93(3):993–1003

    CAS  PubMed  Google Scholar 

  • Barriuso J, Martínez MJ (2015) In silico metagenomes mining to discover novel esterases with industrial application by sequential search strategies. J Microbiol Biotechnol 25(5):732–737

    CAS  PubMed  Google Scholar 

  • Kodzius R, Gojobori T (2015) Marine metagenomics as a source for bioprospecting. Mar Genom 24(Pt 1):21–30

    Google Scholar 

  • Alves L, Silva-Rocha R, Guazzaroni M-E (2017) Enhancing metagenomic approaches through synthetic biology. In: Charles T, Liles M, Sessitsch A (Eds) Functional Metagenomics: Tools and Applications, Springer, New York, pp 75–94

  • Gebresilassie S, Gebretsadik HT, Kabeto K (2011) Preliminary study on geology, mineral potential and characteristics of hot springs from Dallol area, Afar rift, northeastern Ethiopia: Implications for natural resource exploration. Momona Ethiopian J Sci. https://doi.org/10.4314/mejs.v3i2.67710

  • Kotopoulou E et al (2019) A polyextreme hydrothermal system controlled by iron: the case of Dallol at the Afar triangle. ACS Earth Space Chem 3(1):90–99

    CAS  PubMed  Google Scholar 

  • Hamilton TL et al (2011) Biological nitrogen fixation in acidic high-temperature geothermal springs in Yellowstone National Park, Wyoming. Environ Microbiol 13(8):2204–2215

    CAS  PubMed  Google Scholar 

  • Mitchell KR, Takacs-Vesbach CD (2008) A comparison of methods for total community DNA preservation and extraction from various thermal environments. J Ind Microbiol Biotechnol 35(10):1139–1147

    CAS  PubMed  Google Scholar 

  • Emanuelsson BD et al (2015) High-resolution continuous-flow analysis setup for water isotopic measurement from ice cores using laser spectroscopy. Atmos Meas Tech 8(7):2869–2883

    CAS  Google Scholar 

  • Zhou J, Bruns MA, Tiedje JM (1996) DNA recovery from soils of diverse composition. Appl Environ Microbiol 62(2):316–322

    CAS  PubMed  PubMed Central  Google Scholar 

  • Bolger AM, Lohse M, Usadel B (2014) Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30(15):2114–2120

    CAS  PubMed  PubMed Central  Google Scholar 

  • Segata N et al (2012) Metagenomic microbial community profiling using unique clade-specific marker genes. Nat Methods 9(8):811–814

    CAS  PubMed  PubMed Central  Google Scholar 

  • Truong DT et al (2015) MetaPhlAn2 for enhanced metagenomic taxonomic profiling. Nat Methods 12(10):902–903

    CAS  PubMed  Google Scholar 

  • Waterhouse A et al (2018) SWISS-MODEL: homology modelling of protein structures and complexes. Nucleic Acids Res 46(W1):W296-w303

    CAS  PubMed  PubMed Central  Google Scholar 

  • Legendre P, Legendre L (1998) Numerical ecology, 2nd edn. Elsevier, Amsterdam

    Google Scholar 

  • Nurk S et al (2017) metaSPAdes: a new versatile metagenomic assembler. Genome Res 27(5):824–834

    CAS  PubMed  PubMed Central  Google Scholar 

  • Buchfink B, Xie C, Huson DH (2015) Fast and sensitive protein alignment using DIAMOND. Nat Methods 12(1):59–60

    CAS  PubMed  Google Scholar 

  • Bağcı C et al (2019) Introduction to the analysis of environmental sequences: metagenomics with MEGAN. Methods Mol Biol 1910:591–604

    PubMed  Google Scholar 

  • Huson DH et al (2018) MEGAN-LR: new algorithms allow accurate binning and easy interactive exploration of metagenomic long reads and contigs. Biol Direct 13(1):6

    PubMed  PubMed Central  Google Scholar 

  • Huson DH et al (2016) MEGAN Community Edition - Interactive Exploration and Analysis of Large-Scale Microbiome Sequencing Data. PLoS Comput Biol 12(6):e1004957

  • Kanehisa M, Sato Y (2020) KEGG Mapper for inferring cellular functions from protein sequences. Protein Sci 29(1):28–35

    CAS  PubMed  Google Scholar 

  • Gasteiger E et al (2005) Protein Identification and Analysis Tools on the ExPASy Server. In: Walker JM (ed) Humana Press, pp 571–607

  • Geourjon C, Deléage G (1995) SOPMA: significant improvements in protein secondary structure prediction by consensus prediction from multiple alignments. Comput Appl Biosci 11(6):681–684

    CAS  PubMed  Google Scholar 

  • Garnier J, Gibrat JF, Robson B (1996) GOR method for predicting protein secondary structure from amino acid sequence. Methods Enzymol 266:540–553

    CAS  PubMed  Google Scholar 

  • Ghebrezgabher MG, Yang T, Yang X (2016) Long-term trend of climate change and drought assessment in the Horn of Africa. Adv Meteorol 2016:8057641

    Google Scholar 

  • Klotz MG et al (2016) Editorial: systems biology and ecology of microbial mat communities. Front Microbiol 7:115

    PubMed  PubMed Central  Google Scholar 

  • Nobile A et al (2012) Dike-fault interaction during the 2004 Dallol intrusion at the northern edge of the Erta Ale Ridge (Afar, Ethiopia). Geophys Res Lett 39(19).

  • Hallsworth JE et al (2007) Limits of life in MgCl2-containing environments: chaotropicity defines the window. Environ Microbiol 9(3):801–813

    CAS  PubMed  Google Scholar 

  • Herbstein FH, Kapon M, Weissman A (1982) X-ray diffraction as a tool for studying stoichiometry and kinetics of solid state thermal decomposition reactions. Application to the Thermal Decomposition of Bischofite MgCl2 · 6H2O. Israel J Chem 22(3):207–213

  • HCl Leaching and Acid Regeneration (2011) Using MgCl2 Brines and Molten Salt Hydrates, in EPD Congress 2011, pp 520–528

  • Roux S et al (2011) Comparison of 16S rRNA and protein-coding genes as molecular markers for assessing microbial diversity (Bacteria and Archaea) in ecosystems. FEMS Microbiol Ecol 78(3):617–628

    CAS  PubMed  Google Scholar 

  • Poretsky R et al (2014) Strengths and limitations of 16S rRNA gene amplicon sequencing in revealing temporal microbial community dynamics. PLoS ONE 9(4):e93827

    PubMed  PubMed Central  Google Scholar 

  • Ranjan R et al (2016) Analysis of the microbiome: advantages of whole genome shotgun versus 16S amplicon sequencing. Biochem Biophys Res Commun 469(4):967–977

    CAS  PubMed  Google Scholar 

  • Bag S et al (2016) An improved method for high quality metagenomics DNA extraction from human and environmental samples. Sci Rep 6:26775

    CAS  PubMed  PubMed Central  Google Scholar 

  • Ketchum RN et al (2018) DNA Extraction Method Plays a Significant Role When Defining Bacterial Community Composition in the Marine Invertebrate Echinometra mathaei. Front Mar Sci 5(255).

  • Belilla J et al (2019) Hyperdiverse archaea near life limits at the polyextreme geothermal Dallol area. Nat Ecol Evol 3(11):1552–1561

    PubMed  PubMed Central  Google Scholar 

  • Johnson DB et al (2001) Isolation and phylogenetic characterization of acidophilic microorganisms indigenous to acidic drainage waters at an abandoned Norwegian copper mine. Environ Microbiol 3(10):630–637

    CAS  PubMed  Google Scholar 

  • Méndez-García C et al (2015) Microbial diversity and metabolic networks in acid mine drainage habitats. Front Microbiol 6:475

    PubMed  PubMed Central  Google Scholar 

  • Mesa V et al (2017) Bacterial, archaeal, and eukaryotic diversity across distinct microhabitats in an acid mine drainage. Front Microbiol 8:1756

    PubMed  PubMed Central  Google Scholar 

  • Schuler CG, Havig JR, Hamilton TL (2017) Hot spring microbial community composition, morphology, and carbon fixation: implications for interpreting the ancient rock record. Front Earth Sci 5(97)

  • Abreu F et al (2018) Culture-independent characterization of a novel magnetotactic member affiliated to the Beta class of the Proteobacteria phylum from an acidic lagoon. Environ Microbiol 20(7):2615–2624

    CAS  PubMed  Google Scholar 

  • Li, Y., et al., Coupled Carbon, Sulfur, and Nitrogen Cycles Mediated by Microorganisms in the Water Column of a Shallow-Water Hydrothermal Ecosystem. Frontiers in Microbiology, 2018. 9(2718).

  • Widdison, P.E. and T.P. Burt, Nitrogen Cycle, in Encyclopedia of Ecology S.E. Jørgensen and B.D. Fath, Editors. 2008, Academic Press. p. 2526–2533.

  • Vogels GD, Van der Drift C (1976) Degradation of purines and pyrimidines by microorganisms. Bacteriol Rev 40(2):403–468

    CAS  PubMed  PubMed Central  Google Scholar 

  • Kim, B.H. and G.M. Gadd, Degradation of nucleic acid bases, in Bacterial Physiology and Metabolism 2008, Cambridge University Press.: New York. p. 135- 223.

  • Meng L et al (2016) Effects of sucrose amendment on ammonia assimilation during sewage sludge composting. Bioresour Technol 210:160–166

    CAS  PubMed  Google Scholar 

  • Isaji Y et al (2019) Efficient recycling of nutrients in modern and past hypersaline environments. Scientific Reports 9(1):3718

    CAS  PubMed  PubMed Central  Google Scholar 

  • Chen, M.Y., et al., Pseudoxanthomonas taiwanensis sp. nov., a novel thermophilic, N2O-producing species isolated from hot springs. Int J Syst Evol Microbiol, 2002. 52(Pt 6): p. 2155–61.

  • Hedlund, B.P., et al., Potential role of Thermus thermophilus and T. oshimai in high rates of nitrous oxide (N2O) production in ∼80 °C hot springs in the US Great Basin. Geobiology, 2011. 9(6): p. 471–80.

  • Adamczack J et al (2014) NirN protein from Pseudomonas aeruginosa is a novel electron-bifurcating dehydrogenase catalyzing the last step of heme d1 biosynthesis. J Biol Chem 289(44):30753–30762

    CAS  PubMed  PubMed Central  Google Scholar 

  • Ward TW et al (2001) Characterization of the Structural Gene Promoter of Aedes aegypti Densovirus. J Virol 75(3):1325–1331

    CAS  PubMed  PubMed Central  Google Scholar 

  • Dixon R et al (1997) Nif gene transfer and expression in chloroplasts: Prospects and problems. Plant Soil 194(1):193–203

    CAS  Google Scholar 

  • Ivleva NB et al (2016) Expression of Active Subunit of Nitrogenase via Integration into Plant Organelle Genome. PLoS ONE 11(8):e0160951

    PubMed  PubMed Central  Google Scholar 

  • Li XX et al (2016) Using synthetic biology to increase nitrogenase activity. Microb Cell Fact 15:43

    PubMed  PubMed Central  Google Scholar 

  • Burén S, López-Torrejón G, Rubio LM (2018) Extreme bioengineering to meet the nitrogen challenge. Proc Natl Acad Sci U S A 115(36):8849–8851

    PubMed  PubMed Central  Google Scholar 

  • Jones FP et al (2016) Novel European free-living, non-diazotrophic Bradyrhizobium isolates from contrasting soils that lack nodulation and nitrogen fixation genes - a genome comparison. Scientific reports 6:25858–25858

    CAS  PubMed  PubMed Central  Google Scholar 

  • Messer LF et al (2016) High levels of heterogeneity in diazotroph diversity and activity within a putative hotspot for marine nitrogen fixation. Isme j 10(6):1499–1513

    CAS  PubMed  Google Scholar 

  • Lesser MP et al (2018) Diazotroph diversity and nitrogen fixation in the coral Stylophora pistillata from the Great Barrier Reef. Isme j 12(3):813–824

    CAS  PubMed  Google Scholar 

  • McGlynn SE et al (2012) Classifying the metal dependence of uncharacterized nitrogenases. Front Microbiol 3:419

    PubMed  Google Scholar 

  • Pedersen JN et al (2018) Diazotrophs and N(2)-Fixation Associated With Particles in Coastal Estuarine Waters. Front Microbiol 9:2759

    PubMed  PubMed Central  Google Scholar 

  • Pérez CA et al (2017) Biological nitrogen fixation in a post-volcanic chronosequence from south-central Chile. Biogeochemistry 132(1):23–36

    Google Scholar 

  • Black KA, Dos Santos PC (2015) Shared-intermediates in the biosynthesis of thio-cofactors: Mechanism and functions of cysteine desulfurases and sulfur acceptors. Biochim Biophys Acta 1853(6):1470–1480

    CAS  PubMed  Google Scholar 

  • Wang S et al (2013) Transcriptome sequencing of Zhikong scallop (Chlamys farreri) and comparative transcriptomic analysis with Yesso scallop (Patinopecten yessoensis). PLoS ONE 8:e63927

    CAS  PubMed  PubMed Central  Google Scholar 

  • Morett E, Fischer HM, Hennecke H (1991) Influence of oxygen on DNA binding, positive control, and stability of the Bradyrhizobium japonicum NifA regulatory protein. J Bacteriol 173(11):3478–3487

    CAS  PubMed  PubMed Central  Google Scholar 

  • Nonaka A et al (2019) Accessory Proteins of the Nitrogenase Assembly, NifW, NifX/NafY, and NifZ, Are Essential for Diazotrophic Growth in the Nonheterocystous Cyanobacterium Leptolyngbya boryana. Front Microbiol 10:495

    PubMed  PubMed Central  Google Scholar 

  • Koonin EV, Galperin MY, in Sequence - Evolution - Function: Computational Approaches in Comparative Genomics. (2003) Kluwer Academic Copyright © 2003. Kluwer Academic, Boston

    Google Scholar 

  • Poudel, S., et al., Electron Transfer to Nitrogenase in Different Genomic and Metabolic Backgrounds. J Bacteriol, 2018. 200(10).

  • Kiyasu T et al (2000) Contribution of cysteine desulfurase (NifS protein) to the biotin synthase reaction of Escherichia coli. J Bacteriol 182(10):2879–2885

    CAS  PubMed  PubMed Central  Google Scholar 

  • Egener, T., et al., Role of a ferredoxin gene cotranscribed with the nifHDK operon in N(2) fixation and nitrogenase "switch-off" of Azoarcus sp. strain BH72. J Bacteriol, 2001. 183(12): p. 3752–60.

  • Pearson, W.R., An Introduction to Sequence Similarity (“Homology”) Searching. Current Protocols in Bioinformatics, 2013. 42(1): p. 3.1.1–3.1.8.

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Acknowledgements

GMW acknowledges support from the National Institutes of Health 1R01GM132222-01.

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Authors and Affiliations

  1. Institute of Biotechnology, Addis Ababa University, P.O. Box 1176, Addis Ababa, Ethiopia

    Lulit Tilahun & Addis Simachew

  2. School of Earth Sciences, Addis Ababa University, P.O. Box 1176, Addis Ababa, Ethiopia

    Asfawossen Asrat

  3. Department of Molecular and Cell Biology and Biochemistry, Brown University, Box G, 185 Meeting Street, Providence, RI, 02912, USA

    Gary M. Wessel

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  1. Lulit Tilahun
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  2. Asfawossen Asrat
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  3. Gary M. Wessel
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  4. Addis Simachew
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LT designed experiments, collected samples, analyzed data, and wrote drafts of the manuscript. AA, and AS helped designed experiments, collected samples, and edited drafts of the manuscript. GMW helped in DNA isolation, sequencing, and analysis and helped in writing and editing the manuscript.

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Correspondence to Gary M. Wessel.

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Tilahun, L., Asrat, A., Wessel, G.M. et al. In silico determination of nitrogen metabolism in microbes from extreme conditions using metagenomics. Arch Microbiol 203, 2521–2540 (2021). https://doi.org/10.1007/s00203-021-02227-4

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