Zurich Open Repository and Archive University of Zurich Main Library Strickhofstrasse 39 CH-8057 Zurich www.zora.uzh.ch Year: 2014 Ménage-à-trois: The amoeba Nuclearia sp. from Lake Zurich with its ecto- and endosymbiotic bacteria Dirren, Sebastian ; Salcher, Michaela M ; Blom, J F ; Schweikert, M ; Posch, T Abstract: We present a fascinating triad relationship between a eukaryotic amoeba and its two bac- terial symbionts. The morphological characteristics of the amoeba allowed for a confident assignment to the genus Nuclearia (Opisthokonta, Nucleariidae), but species identification resulted in an ambigu- ous result. Sequence analysis indicated an affiliation to the species N. thermophila, however, several morphological features contradict the original description. Amoebal isolates were cultured for several years with their preferred food source, the microcystin-producing harmful cyanobacterium Planktothrix rubescens. Symbioses of the amoeba with ecto- and endosymbiotic bacteria were maintained over this period. Several thousand cells of the ectosymbiont are regularly arranged inside a layer of extracellular polymeric substances produced by the amoeba. The ectosymbiont was identified as Paucibacter tox- inivorans (Betaproteobacteria), which was originally isolated by enrichment with microcystins. We found indications that our isolated ectosymbiont indeed contributed to toxin-degradation. The endosymbiont (Gammaproteobacteria, 15-20 bacteria per amoeba) is enclosed in symbiosomes inside the host cytoplasm and represents probably an obligate symbiont. We propose the name ”Candidatus Endonucleariobacter rarus” for this bacterium that was neither found free-living nor in a symbiotic association. Nucleariidae are uniquely suited model organisms to study the basic principles of symbioses between opisthokonts and prokaryotes. DOI: https://doi.org/10.1016/j.protis.2014.08.004 Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-107199 Journal Article Accepted Version Originally published at: Dirren, Sebastian; Salcher, Michaela M; Blom, J F; Schweikert, M; Posch, T (2014). Ménage-à-trois: The amoeba Nuclearia sp. from Lake Zurich with its ecto- and endosymbiotic bacteria. Protist, 165(5):745- 758. DOI: https://doi.org/10.1016/j.protis.2014.08.004 Manuscript Click here to view linked References Ménage-à-trois: The Amoeba Nuclearia sp. from Lake Zurich with its Ecto- 1 2 3 and Endosymbiotic Bacteria 4 5 a a,b a c 6 Sebastian Dirren , Michaela M. Salcher , Judith F. Blom , Michael Schweikert and Thomas 7 8 Poscha1 9 10 a 11 Limnological Station, Institute of Plant Biology, University of Zurich, Seestrasse 187, CH- 12 13 8802 Kilchberg, Switzerland 14 15 b 16 Biology Centre of the Academy of Sciences of the Czech Republic, Institute of 17 18 Hydrobiology, Na Sádkách 7, CZ-37005 České Budějovice, Czech Republic 19 20 cDepartment of Zoology, Institute of Biology, University of Stuttgart, Pfaffenwaldring 57, 21 22 23 D-70569 Stuttgart, Germany 24 25 26 27 28 Running Title: The Amoeba Nuclearia and its Bacterial Symbionts. 29 30 31 32 33 Number of pages (incl. references): 26 34 35 Number of tables: 2 36 37 Number of figures: 6 38 39 40 Number of Supplementary Figures: 4 (merged in one pdf-file) 41 42 Second revised version, Intended as Original Paper in „Protist‟ 43 44 45 46 47 1Corresponding author: 48 49 PD Dr. Thomas Posch 50 51 Limnological Station, Institute of Plant Biology, University of Zurich 52 53 Seestrasse 187, CH-8802 Kilchberg, Switzerland 54 55 Phone: 0041 44 634 9224 56 Fax: 0041 44 634 9225 57 58 e-mail: [email protected] 59 60 61 62 63 1 64 65 1 1 Abstract 2 3 4 2 We present a fascinating triad relationship between a eukaryotic amoeba and its two bacterial 5 6 3 symbionts. The morphological characteristics of the amoeba allowed for a confident 7 8 9 4 assignment to the genus Nuclearia (Opisthokonta, Nucleraiidae), but species identification 10 11 5 resulted in an ambiguous image. Sequence analysis indicated an affiliation to the species N. 12 13 6 thermophila, 14 however, several morphological features contradict the original description. 15 16 7 Amoebal isolates were cultured for several years with their preferred food source, the 17 18 8 microcystin-producing harmful cyanobacterium Planktothrix rubescens. Symbioses of the 19 20 21 9 amoeba with ecto- and endosymbiotic bacteria were maintained over this period. Several 22 23 10 thousand cells of the ectosymbiont are regularly arranged inside a layer of extracellular 24 25 26 11 polymeric substances produced by the amoeba. It was identified as Paucibacter toxinivorans 27 28 12 (Betaproteobacteria), which was originally isolated by enrichment with microcystins. We 29 30 31 13 found indications that our isolated ectosymbiont indeed contributed to toxin-degradation. The 32 33 14 endosymbiont (Gammaproteobacteria, 15-20 bacteria per amoeba) is enclosed in 34 35 15 symbiosomes inside the host cytoplasm and represents probably an obligate symbiont. We 36 37 38 16 propose the name “Candidatus Endonucleariobacter rarus” for this bacterium that was neither 39 40 17 found free-living nor in a symbiotic association. Nucleariidae are uniquely suited model 41 42 43 18 organisms to study the basic principles of symbioses between opisthokonts and prokaryotes. 44 45 46 19 47 48 49 20 Key words: Bacteria-protist symbioses; Ectosymbionts; Endosymbionts; Feeding; 50 51 52 21 Nucleariidae; Paucibacter toxinivorans. 53 54 55 56 57 58 59 60 61 62 63 2 64 65 22 Introduction 1 2 23 3 Two billion years of co-evolution between eukaryotes and prokaryotes has led to the 4 5 24 establishment of numerous symbiotic associations (Moya et al. 2008). Essentially, all higher 6 7 25 animals and plants live in symbiosis with several, up to thousands of prokaryotic species 8 9 10 26 (Marx 2004; Paracer and Ahmadjian 2000; Ruby et al. 2004). Nevertheless, symbioses with 11 12 27 bacteria are not restricted to multicellular organisms. Bacterivorous protists live in intimate 13 14 15 28 contact with bacteria, and this spatial co-occurrence is a potential playground for novel 16 17 29 symbioses (Görtz and Brigge 1998; Nowack and Melkonian 2010; Schmitz-Esser et al. 2008). 18 19 20 30 Symbiotic bacteria of protists (Gast et al. 2009) are either attached to extracellular structures 21 22 31 (ectosymbionts) or inside the host itself (endosymbionts). 23 24 32 In this article the term symbiosis is used as it was first mentioned in a biological 25 26 27 33 context by Heinrich Anton de Bary in the year 1879 (see Appendix 1 in Paracer and 28 29 34 Ahmadjian (2000)). Symbiosis includes any kind of coevolutionary based „living together‟ of 30 31 32 35 „dissimilar individuals‟. Thus it comprises the whole spectrum of possible interactions 33 34 36 reaching from parasitism to mutualism. 35 36 37 37 So far, only a few associations of bacteria with protists have been characterized in 38 39 38 detail, as symbiotic bacteria are often uncultivable when separated from their hosts. Probably 40 41 39 42 in many cases the appropriate cultivation methods and media for a successful isolation of 43 44 40 symbionts have not been found. However, cultivation outside an obligate host has still to be 45 46 41 considered challenging or even impossible with the available methods. Nowadays, culture- 47 48 49 42 independent techniques like PCR, cloning and sequencing of marker genes, metagenomics, 50 51 43 and fluorescence in situ hybridization (FISH) ease at least the identification of symbiotic 52 53 54 44 microorganisms. 55 56 45 Here we present a triad relationship, in which an amoeboid host is associated with 57 58 Nuclearia, 59 46 endo- as well as ectosymbiotic prokaryotes. The amoeba belongs to the genus the 60 61 47 type genus of the family Nucleariidae, which was originally assigned to the class Filosea by 62 63 3 64 65 48 Cann and Page (1979). Based on molecular phylogenetic analyses, nucleariid amoebae are a 1 2 49 3 sister group of Fungi (Brown et al. 2009) belonging to the diverse group of the Opisthokonta 4 5 50 (Liu et al. 2009; Steenkamp et al. 2006; Zettler et al. 2001). This phylogenetic position makes 6 7 51 them uniquely suited model organisms to study the basic principles of symbiosis between 8 9 10 52 opisthokont eukaryotes and prokaryotes. One typical feature of some Nuclearia species is the 11 12 53 excretion of extracellular polymeric substances (EPS) that form a mucous sheath around the 13 14 15 54 cell - already observed by Artari (1889). A positive periodic acid-schiff (PAS) staining of the 16 17 55 EPS, indicating its polysaccharide character, was shown for N. radians (described as 18 19 20 56 Nucleosphaerium tuckeri by Cann and Page (1979)), for N. delicatula (Cann 1986) and for N. 21 22 57 simplex (Pernin 1976). The EPS can be colonized by ectosymbiotic bacteria that are arranged 23 24 58 regularly a few micrometres from the cell membrane (Cann 1986; Cann and Page 1979; 25 26 27 59 Patterson 1984). So far, endosymbiotic bacteria have been detected in two nucleariid species. 28 29 60 N. radians harbours endosymbionts that are surrounded by a peribacterial membrane in the 30 31 32 61 cytoplasm (Cann and Page 1979), and the amphizoic N. pattersoni has a rickettsial 33 34 62 endosymbiont (Dyková et al. 2003). 35 36 37 63 Besides being already a complex community of three microorganisms, several 38 39 64 nucleariid amoebae have a peculiar feeding behaviour with respect to prey ingestion and 40 41 65 Nuclearia 42 selection (Artari 1889; Cann 1986; Cann and Page 1979). These species can ingest 43 44 66 toxic filamentous cyanobacteria as their sole source of food. The herein presented Nuclearia 45 46 67 species isolated from Lake Zurich (Switzerland) preferentially feeds on the filamentous 47 48 49 68 cyanobacterium Planktothrix rubescens, which is the main primary producer in this 50 51 69 ecosystem (Posch et al.