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A Nitrospira Metagenome Illuminates the Physiology and Evolution of Globally Important Nitrite-Oxidizing Bacteria

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A Nitrospira Metagenome Illuminates the Physiology and Evolution of Globally Important Nitrite-Oxidizing Bacteria A Nitrospira metagenome illuminates the physiology and evolution of globally important nitrite-oxidizing bacteria Sebastian Lückera, Michael Wagnera, Frank Maixnera,1, Eric Pelletierb,c,d, Hanna Kocha, Benoit Vacherieb, Thomas Ratteie, Jaap S. Sinninghe Damstéf, Eva Spieckg, Denis Le Paslierb,c,d, and Holger Daimsa,2 aDepartment of Microbial Ecology, Ecology Centre, University of Vienna, 1090 Vienna, Austria; bCommissariat à l’Energie Atomique, Genoscope, 91000 Evry, France; cCentre National de la Recherche Scientifique, UMR8030, 91000 Evry, France; dUniversité d’Evry-Val-d’Essonne, 91000 Evry, France; eDepartment for Computational Systems Biology, Ecology Centre, University of Vienna, 1090 Vienna, Austria; fDepartment of Marine Organic Biogeochemistry, Royal Netherlands Institute for Sea Research, 1790 AB Den Burg, The Netherlands; and gBiozentrum Klein Flottbek, Mikrobiologie und Biotechnologie, University of Hamburg, 22609 Hamburg, Germany Edited by Edward F. DeLong, Massachusetts Institute of Technology, Cambridge, MA, and approved June 18, 2010 (received for review March 25, 2010) Nitrospira are barely studied and mostly uncultured nitrite-oxidizing ogy and physiology (e.g., 5, 10, 11). Furthermore, except for one bacteria, which are, according to molecular data, among the most 137-kbp contig (12), genomic sequences from Nitrospira have not diverse and widespread nitrifiers in natural ecosystems and biological been obtained yet. This situation has been highly unsatisfactory wastewater treatment. Here, environmental genomics was used to because deeper insight into the biology of these elusive NOB is reconstruct the complete genome of “Candidatus Nitrospira defluvii” crucial for a better understanding of nitrogen cycling in natural and from an activated sludge enrichment culture. On the basis of this engineered systems. first-deciphered Nitrospira genome and of experimental data, we Recently, a Nitrospira strain was enriched from activated show that Ca.N.defluvii differs dramatically from other known sludge and partly characterized (13). This organism, tentatively “ fl ” nitrite oxidizers in the key enzyme nitrite oxidoreductase (NXR), named Candidatus Nitrospira de uvii, belongs to Nitrospira in the composition of the respiratory chain, and in the pathway sublineage I, which is most important for sewage treatment (5) but has no representative in pure culture. Here, the complete used for autotrophic carbon fixation, suggesting multiple indepen- MICROBIOLOGY fl dent evolution of chemolithoautotrophic nitrite oxidation. Adapta- genome of Ca.N.deuvii was reconstructed from a meta- tions of Ca.N.defluvii to substrate-limited conditions include an genomic library of the enrichment. More than two decades after unusual periplasmic NXR, which is constitutively expressed, and Nitrospira were discovered (8), we provide an analysis of pathways for the transport, oxidation, and assimilation of simple a Nitrospira genome with previously unmatched insight into the organic compounds that allow a mixotrophic lifestyle. The reverse biology of Nitrospira, show striking differences in key metabolic pathways between Nitrospira and other NOB, and change the tricarboxylic acid cycle as the pathway for CO2 fixation and the lack − of most classical defense mechanisms against oxidative stress current perception on the evolution of NO2 oxidation. Nitrospira suggest that evolved from microaerophilic or even an- Results and Discussion aerobic ancestors. Unexpectedly, comparative genomic analyses in- Genome Reconstruction. Quantitative FISH has shown that the dicate functionally significant lateral gene-transfer events between − NO -oxidizing enrichment consisted of 86% of Ca.N.defluvii and the genus Nitrospira and anaerobic ammonium-oxidizing plancto- 2 did not contain other known NOB (13). The complete genome of mycetes, which share highly similar forms of NXR and other pro- Ca.N.defluvii was reconstructed from this enrichment by an en- teins reflecting that two key processes of the nitrogen cycle are vironmental genomics approach similar to that used for inferring evolutionarily connected. the genome sequence of the anaerobic ammonium-oxidizing bac- terium (“anammox” organism) “Candidatus Kuenenia stuttgar- environmental genomics | nitrification | Nitrospirae tiensis” (14). The completeness and correct assembly of the Nitrospira genome was indicated by the retrieval of all 63 clusters of itrification, the microbially catalyzed sequential oxidation of orthologous groups (COGs) of proteins, which are present in all Nammonia via nitrite to nitrate, is a key process of the bio- genomes in the current COG database (Fig. S1), by lack of suspi- geochemical nitrogen cycle and of biological wastewater treatment. cious redundancy in gene content, and by the presence of all es- The second step of nitrification is carried out by chemo- sential genes in key biosynthetic pathways. The low frequency of lithoautotrophic nitrite-oxidizing bacteria (NOB), which are phy- single nucleotide polymorphisms (about one per 500 kbp) strongly logenetically heterogeneous (1) and occur in a wide range of aquatic suggests that the enrichment culture contained only one Nitrospira and terrestrial ecosystems. Most studies on the physiology of NOB strain. Key features of the genome are summarized in Table S1 and used pure cultures of Nitrobacter, which belong to the Alphapro- Fig. S1. About 30% of the predicted coding sequences (CDS) have teobacteria (1), and complete genome sequences from NOB are available for three Nitrobacter strains (2, 3) and the marine gam- maproteobacterium Nitrococcus mobilis (GenBank accession no. Author contributions: S.L., M.W., D.L.P., and H.D. designed research; S.L., F.M., H.K., B.V., NZ_AAOF00000000). However, cultivation-independent molec- and J.S.S.D. performed research; T.R. and E.S. contributed new reagents/analytic tools; S.L., ular methods revealed that Nitrospira, forming a deeply branching F.M., E.P., T.R., and H.D. analyzed data; and S.L., M.W., and H.D. wrote the paper. lineage in the bacterial phylum Nitrospirae (4), are by far the most The authors declare no conflict of interest. diverse and abundant NOB (5). In addition to their wide distribu- This article is a PNAS Direct Submission. tion in natural habitats such as soils (6), sediments (7), the oceans Freely available online through the PNAS open access option. (8), and hot springs (9), members of the genus Nitrospira are the Data deposition: The sequence reported in this paper has been depositied in the GenBank predominant NOB in wastewater treatment plants (5) and thus database (accession no. FP929003). belong to the microorganisms most relevant for biotechnology. 1Present address: Institute for Mummies and the Iceman, EURAC Research, Viale Druso 1, The immense ecological and technical significance of Nitrospira 39100 Bolzano, Italy. contrasts with our scarce knowledge about these bacteria. As the 2To whom correspondence should be addressed. E-mail: [email protected]. majority of Nitrospira are uncultured, and the available cultures are This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. difficult to maintain, only a few studies have addressed their ecol- 1073/pnas.1003860107/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1003860107 PNAS Early Edition | 1of6 Downloaded by guest on October 1, 2021 no homologs in other organisms, reflecting the distant relationship and D). Homologous [Fe-S] clusters mediate intramolecular of Nitrospira to other bacteria and the lack of genome sequences electron transfer in nitrate reductase A of Escherichia coli (24). from the genus Nitrospira in public databases. Furthermore, only All NxrA and NxrB copies of Ca.N.defluvii lack trans- two lineages within the phylum Nitrospirae have been explored on membrane helices, although NXR is membrane-associated in a genomic level. The closest genome-sequenced relatives of Ca.N. Nitrospira (21). Theoretically, the α/β-complex might cluster with defluvii belong to the genus Leptospirillum and are aerobic acido- a membrane-bound terminal oxidase that receives electrons from philic iron oxidizers (15–17). In addition, the genome sequence of NXR. However, other enzymes in the DMSO reductase family the anaerobic sulfate reducer Thermodesulfovibrio yellowstonii contain an additional membrane-integral γ-subunit, which is the (GenBank accession no. NC_011296), also belonging to the membrane anchor of the holoenzyme and channels electrons Nitrospirae, is publicly available. between the β-subunit and the electron transport chain via one or two hemes (25). Four proteins encoded by Ca.N.defluvii − Nitrite Oxidation and Energy Metabolism. The key enzyme for NO2 could be heme-containing subunits of NXR (Table S2). Each has oxidation by NOB is nitrite oxidoreductase (NXR), which shuttles one transmembrane domain and an N-terminal signal peptide − two electrons per oxidized NO2 into the electron transport chain. for translocation via the Sec pathway. The largest candidate In Nitrobacter, NXR is an iron-sulfur molybdoprotein (18) located at (66.7 kDa) is a c-type cytochrome with two predicted heme- the inner cell membrane and at the intracytoplasmic membranes binding sites. The other three proteins are smaller (29.7–34.3 (ICM). The reaction catalyzed by this NXR is reversible, so that the kDa) and remotely similar to the γ-subunit of chlorate reductase, − enzyme also reduces NO3 with electrons derived from organic which contains one b-type heme (26). These genes are not in compounds. Depending
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