1 Abundant toxin-related genes in the genomes of beneficial symbionts 2 from deep-sea hydrothermal vent mussels 3 4 Lizbeth Sayavedra1, Manuel Kleiner1,*, Ruby Ponnudurai2,*, Silke Wetzel1, Eric 5 Pelletier3,4,5, Valerie Barbe3, Nori Satoh6, Eiichi Shoguchi6, Dennis Fink1, 6 Corinna Breusing7, Thorsten B.H. Reusch7, Philip Rosenstiel8, Markus B. 7 Schilhabel8, Dörte Becher9,10, Thomas Schweder2;9, Stephanie Markert2,9, 8 Nicole Dubilier1,11, Jillian M. Petersen1#& 9 1Max Planck Institute for Marine Microbiology, Celsiusstrasse 1, 28359 10 Bremen, Germany 11 2Institute of Pharmacy, Ernst-Moritz-Arndt-University, Felix-Hausdorff-Strasse 12 3, 17487 Greifswald, Germany 13 3CEA - Genoscope, 2 rue Gaston Crémieux, 91000 Evry, France 14 4CNRS UMR8030, 2 rue Gaston Crémieux, 91000 Evry, France 15 5Université d'Evry Val d'Essonne, 2 rue Gaston Crémieux, 91000 Evry, 16 France 17 6Marine Genomics Unit, Okinawa Institute of Science and Technology 18 Graduate University, Onna, Okinawa 904-0495, Japan 19 7GEOMAR Helmholtz Centre for Ocean Research Kiel, Evolutionary Ecology, 20 Düsternbrooker Weg 20, 24105 Kiel, Germany 21 8Institute of Clinical Molecular Biology (IKMB), Schittenhelmstr. 12 22 24105 Kiel, Germany 1 23 9Institute of Marine Biotechnology, Walther-Rathenau-Strasse 49a, 17489 24 Greifswald, Germany 25 10Institute of Microbiology, Ernst-Moritz-Arndt-University, Friedrich-Ludwig- 26 Jahn-Strasse 15, 17487 Greifswald, Germany 27 11University of Bremen, 28359 Bremen, Germany 28 *Equal contribution 29 #Corresponding author – Jillian M. Petersen 30 Max Planck Institute for Marine Microbiology 31 Celsiusstrasse 1 32 28359 Bremen 33 Germany 34 Tel: +49 421 2028 823 35 Email: [email protected] 36 &Current address 37 Department of Microbiology and Ecosystem Science 38 Division of Microbial Ecology 39 Research Network Chemistry Meets Microbiology 40 University of Vienna 41 Althanstrasse 14 42 1090 Vienna 43 Austria 2 44 Abstract 45 Bathymodiolus mussels live in symbiosis with intracellular sulfur-oxidizing 46 (SOX) bacteria that provide them with nutrition. We sequenced the SOX 47 symbiont genomes from two Bathymodiolus species. Comparison of these 48 symbiont genomes with those of their closest relatives revealed that the 49 symbionts have undergone genome rearrangements, and up to 35% of their 50 genes may have been acquired by horizontal gene transfer. Many of the 51 genes specific to the symbionts were homologs of virulence genes. We 52 discovered an abundant and diverse array of genes similar to insecticidal 53 toxins of nematode and aphid symbionts, and toxins of pathogens such as 54 Yersinia and Vibrio. Transcriptomics and proteomics revealed that the SOX 55 symbionts express the toxin-related genes (TRGs) in their hosts. We 56 hypothesize that the symbionts use these TRGs in beneficial interactions with 57 their host, including protection against parasites. This would explain why a 58 mutualistic symbiont would contain such a remarkable ‘arsenal’ of TRGs. 3 59 Introduction 60 Mussels of the genus Bathymodiolus dominate deep-sea hydrothermal vents 61 and cold seeps worldwide. The key to their ecological and evolutionary 62 success is their symbiosis with chemosynthetic bacteria that provide them 63 with nutrition (von Cosel, 2002; Van Dover, 2002). Bathymodiolus mussels 64 host their symbionts inside specialized gill epithelial cells called bacteriocytes 65 (Cavanaugh et al., 2006; Petersen and Dubilier, 2009). 66 Their filtering activity exposes Bathymodiolus mussels to a plethora of diverse 67 microbes in their environment. Despite this, they are colonized by only one or 68 a few specific types of chemosynthetic symbionts. Some mussel species 69 associate exclusively with sulfur-oxidizing (SOX) symbionts that use reduced 70 sulfur compounds and sometimes hydrogen as an energy source, and carbon 71 dioxide as a carbon source. Some have only methane-oxidizing (MOX) 72 symbionts that use methane as an energy source and carbon source. Some 73 mussel species host both types in a dual symbiosis (Distel et al., 1995; 74 Dubilier et al., 2008; Duperron et al., 2006; Fisher et al., 1993; Petersen et al., 75 2011). In all species except one, a single 16S rRNA phylotype for each type of 76 symbiont (SOX or MOX) is found in the gills (Dubilier et al., 2008). There are 77 more than 30 described Bathymodiolus species, and most associate with a 78 characteristic symbiont phylotype which is not found in other species 79 (Duperron et al., 2013). 80 Although these associations are clearly very specific, the molecular 81 mechanisms that underpin this specificity are still unknown. No 82 chemosynthetic symbiont has ever been obtained in pure culture. Therefore, 4 83 molecular methods for investigating uncultured microbes have been essential 84 for understanding their biodiversity, function, and evolution (reviewed by 85 Dubilier et al., 2008). 86 The Bathymodiolus symbionts are assumed to be horizontally transmitted, 87 which means that each new host generation must take up their symbionts 88 from the surrounding environment or co-occurring adults (DeChaine et al., 89 2006; Fontanez and Cavanaugh, 2014; Kadar et al., 2005; Wentrup et al., 90 2014; Won et al., 2003b). To initiate the symbiosis, hosts and symbionts must 91 have evolved highly specific recognition and attachment mechanisms. Once 92 they have been recognized, the symbionts need to enter host cells and avoid 93 immediate digestion, just like other intracellular symbionts such as 94 Burkholderia rhizoxinica and Rhizobium leguminosarum, or pathogens such 95 as Legionella, Listeria or Yersinia (Hentschel et al., 2000; Moebius et al., 96 2014). Indeed, like many intracellular pathogens, the Bathymodiolus 97 symbionts seem to induce a loss of microvilli on the cells they colonize 98 (Bhavsar et al., 2007; Cossart and Sansonetti, 2004; Haglund and Welch, 99 2011; Wentrup et al., 2014). Finally, the symbionts achieve dense populations 100 inside the host cells (e.g. Duperron et al., 2006; Halary et al., 2008). 101 Therefore, they must be able to avoid immediate digestion by their hosts. 102 Although the mechanisms of host cell entry and immune evasion have been 103 extensively studied in pathogens and plant-microbe associations such as the 104 Rhizobia-legume symbiosis, far less is known about the mechanisms 105 beneficial symbionts use to enter and survive within animal host cells. 106 The symbiosis between Vibrio fisheri bacteria and Euprymna scolopes squid 107 is one of the few beneficial host-microbe associations where the molecular 5 108 mechanisms of host-symbiont interaction have been investigated. A number 109 of factors are involved in initiating this symbiosis such as the symbiont- 110 encoded ‘TCT toxin’, which is related to the tracheal cytotoxin of Bordetella 111 pertussis (McFall-Ngai et al., 2013). A few studies of intracellular insect 112 symbionts have shown that they use type III and type IV secretion systems to 113 establish and maintain their association with their host (reviewed by Dale and 114 Moran, 2006; Snyder and Rio, 2013). These secretion systems are commonly 115 used by intracellular pathogens to hijack host cell processes, allowing their 116 entry and survival within host cells (e.g. Backert and Meyer, 2006; Hueck, 117 1998; Steele-Mortimer et al., 2002). An example is the Sodalis symbionts of 118 aphids and weevils, which use a type III secretion system for entry to the host 119 cell and are thought to have evolved from pathogens (Clayton et al., 2012; 120 Dale et al., 2001). The virulence determinants of their pathogenic ancestors 121 might therefore have been co-opted for use in beneficial interactions with their 122 insect hosts. 123 In contrast to the Sodalis symbionts of insects and the Vibrio symbionts of 124 squid, the Bathymodiolus SOX symbionts are not closely related to any known 125 pathogens. Moreover, because they fall interspersed between free-living 126 sulfur-oxidizing bacteria in 16S rRNA phylogenies, they are hypothesized to 127 have evolved multiple times from free-living ancestors (Figure 2-figure 128 supplement 1) (Petersen et al., 2012). Comparative genomics is a powerful 129 tool for identifying the genomic basis of beneficial and pathogenic interactions, 130 particularly if the symbionts or pathogens have close free-living relatives that 131 do not associate with a host (e.g. Galagan, 2014; Ogier et al., 2014; Zuleta et 132 al., 2014). Genomes of closely-related free-living and symbiotic relatives of 6 133 Bathymodiolus SOX symbionts were recently published. Their closest free- 134 living relatives are marine sulfur-oxidizing bacteria called SUP05, which are 135 abundant in the world’s oceans, particularly in oxygen minimum zones and 136 hydrothermal plumes (Anantharaman et al., 2012; Lavik et al., 2009; Petersen 137 and Dubilier, 2014; Sunamura et al., 2004; Walsh et al., 2009; Wright et al., 138 2012). The Bathymodiolus SOX symbionts and SUP05 bacteria form a 139 monophyletic clade together with the sulfur-oxidizing symbionts of vesicomyid 140 clams based on 16S rRNA gene phylogenies (Figure 2-figure supplement 1) 141 (Distel et al., 1995; Petersen et al., 2012). Closed genomes are available for 142 the symbionts of two clam species (Kuwahara et al., 2007; Newton et al., 143 2007). 144 All members of this monophyletic group (the mussel and clam symbionts, and 145 SUP05) share similar core metabolic features. They are all capable of 146 autotrophic growth, and all use reduced sulfur compounds as an energy 147 source (Newton et al., 2008; Walsh et al., 2009). They can differ in auxiliary 148 metabolic capabilities such as hydrogen oxidation, nitrate reduction or 149 mixotrophy (Anantharaman et al., 2012; Murillo et al., 2014; Newton et al., 150 2008; Petersen et al., 2011). However, the major difference between these 151 organisms is their lifestyle: SUP05 bacteria are exclusively free-living. The 152 clam symbionts are exclusively host-associated, are vertically transmitted, 153 and have reduced genomes. The Bathymodiolus symbionts appear to have 154 adapted to both niches, as they have a host-associated stage and are 155 assumed to also have a free-living stage.