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Environmental Breviatea harbour mutualistic Arcobacter epibionts

Abstract

Breviatea form a lineage of free living, unicellular protists, distantly related to animals and fungi1,2. This lineage emerged almost one billion years ago, when the oceanic oxygen content was low, and extant Breviatea have evolved or retained an anaerobic lifestyle3,4. Here we report the cultivation of Lenisia limosa, gen. et sp. nov., a newly discovered breviate colonized by relatives of animal-associated Arcobacter. Physiological experiments show that the association of L. limosa with Arcobacter is driven by the transfer of hydrogen and is mutualistic, providing benefits to both partners. With whole-genome sequencing and differential proteomics, we show that an experimentally observed fitness gain of L. limosa could be explained by the activity of a so far unknown type of NAD(P)H-accepting hydrogenase, which is expressed in the presence, but not in the absence, of Arcobacter. Differential proteomics further reveal that the presence of Lenisia stimulates expression of known ‘virulence’ factors by Arcobacter. These proteins typically enable colonization of animal cells during infection5, but may in the present case act for mutual benefit. Finally, re-investigation of two currently available transcriptomic data sets of other Breviatea4 reveals the presence and activity of related hydrogen-consuming Arcobacter, indicating that mutualistic interaction between these two groups of microbes might be pervasive. Our results support the notion that molecular mechanisms involved in virulence can also support mutualism6, as shown here for Arcobacter and Breviatea.

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Figure 1: Relatives of animal-associated Arcobacter colonize Breviatea.
Figure 2: Symbiotic metabolism of L. limosa and Arcobacter.
Figure 3: The fitness of L. limosa depends on its symbiont.

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Acknowledgements

We thank T. Hargesheimer, D. Liu, G. Klockgether, P. Hach, R. Appel and I. Kattelmann for technical assistance, A. Simpson, G. Strous and Fulvio Reggiori for comments on electron micrographs, and C. Hubert, E. Ruff, S. Ahmerkamp and N. Dubilier for discussions. This study was supported by European Research Council starting grant MASEM 242635 (M.S., E.H., J.C.), the Campus Alberta Innovation Chair Program (M.S., E.H., X.D.), the Canadian Foundation for Innovation (M.S), the Alberta Small Equipment Grant Program (M.S.), the German Federal State Nordrhein-Westfalen (M.S.), the Max Planck Society, and the Natural Sciences and Engineering Research Council of Canada for a Banting fellowship to M.K. and a Discovery Grant to M.S.

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Authors

Contributions

E.H. and J.C. performed sampling, cultivation and physiological experiments. M.K. performed proteomics and data analysis. D.R. performed transmission electron microscopy. S.L. and E.H. performed scanning electron microscopy. E.H. performed CARD-FISH imaging. H.T. performed next-generation sequencing, H.G.-V. performed read processing, assembly and binning. E.H. performed in silico processing of next-generation sequencing data with assistance from H.G.-V., X.D. and M.S. M.W.B., C.W.S. and A.J.R. analysed sequences for Arcobacter associated with S. tetraspora. E.H., B.V. and K.B performed chemical analysis with input from J.M, K.-U.H. and M.S. The experimental design was developed jointly by M.S., E.H., J.M., M.K. and K.-U.H. E.H. wrote the manuscript with input from all co-authors.

Corresponding authors

Correspondence to Emmo Hamann or Marc Strous.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Micrographs for L. limosa and epibiotic Arcobacter.

a, Scanning electron micrograph showing L. limosa and associated bacteria. Pilus (1) connecting Arcobacter (5) with L. limosa. Pseudopodial extensions (2) are used for the acquisition of prey bacteria (3) (Alteromonas). Short anterior flagellum (4). Long posterior flagellum (6). b–e, CARD-FISH labelling with probes targeting the SSU rRNA of L. limosa (Euk516 in red) and Arcobacter (Epsy914 in green). The scale bar applies to all figures. fi, CARD-FISH labelling of L. limosa with probes targeting the SSU rRNA of A. macleodii (Alt184 in green). The scale bar applies to all figures. jr, Transmission electron micrographs showing different structural features of L. limosa. Mitochondria-related organelle (mro), nucleus (nucl), digestive vacuoles (dv), double basal body (bb), endoplasmic reticulum (er), inner (im) and outer membrane (om), tubular cristae (cristae), extracellular matrix (ex), bacterium (bac), membrane (mem), flagellum (flag), multivesicular body (mb). For a–i, each specimen shown represents at least ten specimens for which images were recorded.

Extended Data Figure 2 Relative abundance of L. limosa and co-enriched bacteria under different growth conditions.

The abundance of L. limosa and its associated microbiota was determined at three different conditions (treatments) with three independent experiments per treatment: 1–3, presence of nitrous oxide and prey bacteria; 4–6, absence of nitrous oxide and presence of prey bacteria; 7–9, presence of nitrous oxide, dissolved organic nutrients and hydrogen and absence of prey bacteria. Relative abundances were determined via proteomics and estimated on the basis of the total normalized spectrum count per population.

Extended Data Figure 3 Genome statistics for L. limosa and epibiotic Arcobacter.

The pie chart represents the classifications of gene models into functional categories for Arcobacter. Gene classifications were performed with the RAST functional annotations and the SEED subsystem database32.

Extended Data Figure 4 A new type of NAD(P)H-dependent Fe-hydrogenase.

The genome of L. limosa encoded a so far undescribed NAD(P)H-dependent Fe-hydrogenase. Genes with identical domain architecture were also identified in P. biforma and T. vaginalis (shown in bold type). The scale bars represent substitution rate per site. a, Phylogeny of the Fe-hydrogenases domain. b, Phylogeny of the NAD/NADP binding domain. Phylogenies were inferred by RAxML using the WAG amino-acid replacement matrix. c, Domain architecture of the NAD(P)H-dependent Fe-hydrogenase (2) compared with the domain architecture of Fe-hydrogenase (3) and the NADPH accepting domain of the cyt P450 reductase (1). The scale bar shows approximate amino-acid positions. d, Predicted electron flow within the NAD(P)H-dependent Fe-hydrogenase indicates the capability for a proton-dependent recycling of NAD(P)H. Note: the shape of the model does not intent to depict the actual three-dimensional structure of the protein.

Extended Data Figure 5 Maximum likelihood tree of quinone-reactive Ni/Fe-hydrogenases (subunit hydB).

The tree shows the phylogenetic relation of quinone-reactive Ni/Fe-hydrogenases from Arcobacter associated with S. tetraspora, P. biforma and L. limosa (indicated in red). Circles represent bootstrap support values for each node. The scale bar represents substitution rate per site.

Extended Data Figure 6 The fitness of L. limosa depends on its symbiont.

Syntrophy was enabled by the presence of nitrous oxide acting as electron acceptor for bacterial hydrogen oxidation. a, Inhibition of nitrous oxide reduction (addition of the competitive inhibitor acetylene, see arrow) led to a reduced growth of L. limosa and reduced respiration rates. To monitor respiration rates, 13C-enriched Alteromonas were added together with acetylene. Digestion of 13C-labelled bacteria by L. limosa led to the production of 13C-bicarbonate, which was measured after conversion to 13CO2 (right). Similar effects on the growth and respiration rates were observed after adding hydrogen (b) or hydrogen and acetate (c) to a culture. d, Growth of L. limosa and production of hydrogen and fatty acids while growing syntrophically (nitrous oxide present). e, Growth of L. limosa in the presence of antibacterial antibiotics (nitrous oxide absent). f, Growth of L. limosa in the presence of antibacterial antibiotics (nitrous oxide present). g, Growth of L. limosa in the presence of nitrate (2 mM) and oxygen (0.2 mM). Growth of L. limosa was compared with a culture that contained nitrous oxide (2.2 mM) and with a control culture that did not contain an electron acceptor for hydrogen oxidation. Each panel shows the results of at least five independent experiments, with cell numbers depicted as averages of seven cell counts per experiment; error bars, s.d.

Extended Data Figure 7 Expression levels for Arcobacter proteins involved in attachment and chemotaxis.

a. Expression level of proteins involved in attachment in the presence (red) and absence of L. limosa (blue). b, Expression level of proteins involved in chemotaxis. Expression levels were measured and averaged for three independent experiments per treatment (see also Extended Data Fig. 2). Error bars, s.d. See Supplementary Table 1 for gene accession numbers and statistical tests.

Extended Data Figure 8 Domain architecture of L. limosa fibronectin type III domain-containing proteins.

Protein architectures and conserved protein domains were identified using the SMART protein domain detection tools. See Supplementary Table 1 for gene accession numbers and expression levels.

Extended Data Table 1 Potential presence of Breviatea and Breviatea-associated Arcobacter detected in currently available shotgun metagenomes from marine sediments

Supplementary information

Supplementary Table 1

Accession numbers and per gene expression levels as determined with proteomics. (XLS 273 kb)

Supplementary Table 2

Calculation of the thermodynamic and kinetic feasibility of hydrogen transfer between L. limosa and Arcobacter. (XLS 10 kb)

Supplementary Information

This file contains Supplementary Notes regarding the diagnosis of Lenisia limosa gen. et sp. Nov (PDF 80 kb)

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Hamann, E., Gruber-Vodicka, H., Kleiner, M. et al. Environmental Breviatea harbour mutualistic Arcobacter epibionts. Nature 534, 254–258 (2016). https://doi.org/10.1038/nature18297

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