The ISME Journal (2014) 8, 40–51 & 2014 International Society for Microbial Ecology All rights reserved 1751-7362/14 www.nature.com/ismej ORIGINAL ARTICLE Allying with armored snails: the complete genome of gammaproteobacterial endosymbiont Satoshi Nakagawa1,2, Shigeru Shimamura2, Yoshihiro Takaki3, Yohey Suzuki4, Shun-ichi Murakami1, Tamaki Watanabe1, So Fujiyoshi1, Sayaka Mino1, Tomoo Sawabe1, Takahiro Maeda5,6, Hiroko Makita2, Suguru Nemoto7, Shin-Ichiro Nishimura5,8, Hiromi Watanabe9, Tomo-o Watsuji2 and Ken Takai2 1Laboratory of Microbiology, Faculty of Fisheries Sciences, Hokkaido University, Hakodate, Japan; 2Subsurface Geobiology Advanced Research (SUGAR) Project, Institute of Biogeosciences, Japan Agency for Marine-Earth Science and Technology (JAMSTEC), Yokosuka, Japan; 3Cooperative Research Project for Extremophiles, Institute of Biogeosciences, JAMSTEC, Yokosuka, Japan; 4Department of Earth and Planetary Science, Graduate School of Science, University of Tokyo, Tokyo, Japan; 5Laboratory of Advanced Chemical Biology, Graduate School of Life Science, Hokkaido University, Sapporo, Japan; 6Amino Up Chemical Company, Sapporo, Japan; 7Enoshima Aquarium, Fujisawa, Japan; 8Medicinal Chemistry Pharmaceuticals, Co. Ltd., Sapporo, Japan and 9Marine Biodiversity Research Program, JAMSTEC, Yokosuka, Japan Deep-sea vents harbor dense populations of various animals that have their specific symbiotic bacteria. Scaly-foot gastropods, which are snails with mineralized scales covering the sides of its foot, have a gammaproteobacterial endosymbiont in their enlarged esophageal glands and diverse epibionts on the surface of their scales. In this study, we report the complete genome sequencing of gammaproteobacterial endosymbiont. The endosymbiont genome displays features consistent with ongoing genome reduction such as large proportions of pseudogenes and insertion elements. The genome encodes functions commonly found in deep-sea vent chemoautotrophs such as sulfur oxidation and carbon fixation. Stable carbon isotope (13C)-labeling experiments confirmed the endosymbiont chemoautotrophy. The genome also includes an intact hydrogenase gene cluster that potentially has been horizontally transferred from phylogenetically distant bacteria. Notable findings include the presence and transcription of genes for flagellar assembly, through which proteins are potentially exported from bacterium to the host. Symbionts of snail individuals exhibited extreme genetic homogeneity, showing only two synonymous changes in 19 different genes (13 810 positions in total) determined for 32 individual gastropods collected from a single colony at one time. The extremely low genetic individuality in endosymbionts probably reflects that the stringent symbiont selection by host prevents the random genetic drift in the small population of horizontally transmitted symbiont. This study is the first complete genome analysis of gastropod endosymbiont and offers an opportunity to study genome evolution in a recently evolved endosymbiont. The ISME Journal (2014) 8, 40–51; doi:10.1038/ismej.2013.131; published online 8 August 2013 Subject Category: Microbe-microbe and microbe-host interactions Keywords: chemoautotrophy; deep-sea hydrothermal vent; symbiosis; scaly-foot gastropod Introduction in the Kairei hydrothermal field in the Indian Ocean. Dark-colored sclerites cover the sides of the Scaly-foot gastropods, tentatively named ‘Crysomal- snails’ foot in a roof-tile fashion (Ware´n et al., 2003; lon squamiferum’, were first described in 2001 Goffredi et al., 2004). The dominant crystalline (Van Dover et al., 2001). These snails live a mineral phase is pyrite, whereas greigite, the sulfide sedentary life at the base of a black smoker chimney, analog of magnetite, is present in lower proportions and is responsible for the ferrimagnetism (Ware´n et al., 2003; Suzuki et al., 2006). In addition, the Correspondence: S Nakagawa, Laboratory of Microbiology, shell of scaly-foot gastropod is fortified by a unique Faculty of Fisheries Sciences, Hokkaido University, 3-1-1 multilayer structure (Yao et al., 2010). These Minato-cho, Hakodate 041-8611, Japan. E-mail: [email protected] defenses are probably acquired for the protection Received 5 March 2013; revised 26 June 2013; accepted 1 July against environmental threats including grazing 2013; published online 8 August 2013 (Suzuki et al., 2006). The scaly-foot gastropods, Microbial symbiosis in scaly-foot gastropod S Nakagawa et al 41 however, share the habitat around the base of a black have not been clarified yet. Several genetic and smoker chimney with a different kind of gastropod, biochemical studies demonstrated that the Alviniconcha sp., which have a bare foot and very chemoautotrophic Gammaproteobacteria and thin soft shell. Epsilonproteobacteria fix CO2 via Calvin-Benson- Many deep-sea vent endemic mollusks harbor Basham cycle and reductive tricarboxylic acid specific symbionts, although Alviniconcha gastro- cycle, respectively (Hu¨ gler et al., 2005; Takai et al., pods exhibit marked diversity in their symbiont 2005; Nakagawa and Takai, 2008). In addition, ribotypes within and among populations (Suzuki repertoire of the sulfur-oxidation pathways differs et al., 2005; Beinart et al., 2012). The scaly-foot between deep-sea vent Gammaproteobacteria and gastropod houses its gammaproteobacterial sym- Epsilonproteobacteria (Nakagawa and Takai, 2008; bionts within the cells of its enlarged esophageal Yamamoto and Takai, 2011). However, little is gland (Goffredi et al., 2004). The endosymbiont known about how these differences influence their population is highly clonal within and among snail ecological performance. individuals at the 16S rRNA gene sequence level At present, no genome sequence is available for (Goffredi et al., 2004). In addition, Deltaproteobacteria any gastropod endosymbionts. In this study, and Epsilonproteobacteria densely colonize the we describe the complete genome sequence analysis surface of scales (Goffredi et al., 2004). The of gammaproteobacterial endosymbiont found gastropod nutritionally depends on the intracellular in scaly-foot gastropod. To assess the physiology of gammaproteobacterium that synthesize organics gastropod, we also performed the 13C tracer experi- from CO2 potentially by oxidizing reduced sulfur ments and hemolymph glycan analysis. We compounds (Goffredi et al., 2004). Until recently, sequenced the symbiont genome specifically as an ecophysiological characteristics of symbiotic Gam- effort to address (1) genomic differences with other maproteobacteria in deep-sea vents had remained chemoautotrophs, (2) genome evolution in a mostly unknown because of their resistance to recently derived symbiont and (3) endosymbiont cultivation outside of specialized host tissues. physiology. This study represents a step toward a However, genome sequences have been determined better understanding of genome dynamics and for endosymbionts of Riftia pachyptila (tubeworm) evolution of symbiotic bacteria in deep-sea vents, (Markert et al., 2007) and Calyptogena species and will lead to more specifically focused biochemical (bivalves endemic in deep-sea vents) (Kuwahara investigations. et al., 2007; Newton et al., 2007). Complete genome sequence has also been determined for the sulfur- Materials and methods oxidizing endosymbiont of Olavius algarvensis (shallow oligochaete) (Woyke et al., 2006). These Sampling and DNA/RNA preparation genome sequences have provided new insights into Samples used in this study were obtained by means symbiont evolution, physiological functions and of DSV Shinkai 6500 at the Kairei hydrothermal interactions with their host animals. For example, field (Van Dover et al., 2001) in the Central Indian the marine endosymbionts, including heritable Ridge (251 19.230S, 701 02.420E). All samples ones, have significantly larger genomes with higher were collected from a single colony during dive GC contents compared with insect endosymbionts, no. 1169 on 6 November 2009, unless noted suggesting their recent association with hosts otherwise. Although Alviniconcha sp. represents (Woyke et al., 2006; Kuwahara et al., 2007, Markert the gastropods visually observed from the submer- et al., 2007; Newton et al., 2007). As expected from sible’s window in this hydrothermal field, we the large genome sizes, the marine gammaproteo- discovered a large and dense population of scaly- bacterial endosymbionts retain metabolically foot gastropods covered by Rimicaris shrimps complete genomes, and even have multiple path- (water depth ¼ 2420 m) (Supplementary Figure S1). ways both for energy metabolism (Woyke et al., Once onboard the ship, the esophageal gland was 2006; Kuwahara et al., 2007; Newton et al., 2007) dissected from a single snail (Supplementary Figure and for carbon fixation (Markert et al., 2007). These S1c) and homogenized with glass Teflon homogeni- endosymbiont genomes therefore provide a rare zer powered by a standard electric drill in 5 ml of opportunity to study the early stages of genome phosphate-buffered saline at pH 7.0 on ice. Debris reduction during symbiont evolution. was pelleted at 800 Â g for 5 min, and supernatant In deep-sea vent ecosystems, chemoautotrophic was filtered through a 10-mm nylon mesh filter Epsilonproteobacteria are dominant and metaboli- (Millipore, Billerica, MA, USA). The filtrate was cally versatile, suggesting that deep-sea vent further centrifuged at 14 000 r.p.m. for 10 min chemoautotrophs, even if they are epi- or endosym- to recover