Review TRENDS in Microbiology Vol.13 No.9 September 2005 Chemosynthetic endosymbioses: adaptations to oxic–anoxic interfaces Frank J. Stewart, Irene L.G. Newton and Colleen M. Cavanaugh Department of Organismic and Evolutionary Biology, Harvard University, Cambridge, MA 02138, USA Chemosynthetic endosymbioses occur ubiquitously at hydrothermal vents, where reduced chemicals in anoxic oxic–anoxic interfaces in marine environments. In these vent effluent mix with oxygenated seawater, chemo- mutualisms, bacteria living directly within the cell of a synthetic bacteria flourish. eukaryotic host oxidize reduced chemicals (sulfur or Although free-living chemosynthetic prokaryotes serve methane), fueling their own energetic and biosynthetic as the base of the food chain for some vent organisms, needs, in addition to those of their host. In habitats such reliance on symbiotic chemosynthetic bacteria is the as deep-sea hydrothermal vents, chemosynthetic sym- primary nutritional strategy for many vent invertebrates bioses dominate the biomass, contributing substantially [5]. Both partners are inferred to benefit nutritionally to primary production. Although these symbionts have from these symbioses [6,7]. The invertebrate host facili- yet to be cultured, physiological, biochemical and tates access to the substrates (e.g. sulfide or methane, molecular approaches have provided insights into oxygen, CO2) that are necessary for chemosynthetic symbiont genetics and metabolism, as well as into metabolism (Figure 1). In exchange, the bacterial symbiont–host interactions, adaptations and ecology. symbionts fix carbon that supports the growth and Recent studies of endosymbiont biology are reviewed, maintenance of host biomass. Given the reciprocal with emphasis on a conceptual model of thioauto- benefits to the partners of these associations, it is not trophic metabolism and studies linking symbiont physi- surprising that chemosynthetic symbioses occur widely in ology with the geochemical environment. We also nature. Indeed, following the discovery of symbiont- discuss current and future research directions, focusing dominated vent communities, a variety of other environ- on the use of genome analyses to reveal mechanisms ments characterized by the mixing of oxic and anoxic that initiate and sustain the symbiont–host interaction. zones (e.g. hydrocarbon cold seeps, coastal sediments, mud volcanoes, whale falls) were shown to support chemosyn- thetic symbioses. In all of these environments, symbiosis can be viewed as a bacterial adaptation to simultaneously Introduction sequester reduced energy substrates (sulfur or methane) Symbioses between bacteria and eukaryotes impact the and oxygen by living inside a eukaryotic host. Primary physiology, ecology and evolution of all organisms. Indeed, production by chemosynthetic symbionts in these habitats there is growing consensus that the mitochondria and provides a direct link between symbiosis and biogeochemi- chloroplasts of eukaryotes arose from bacteria that became cal cycling of carbon and energy substrates (e.g. sulfur). established intracellularly in primitive single-celled organ- Here we review recent studies of endosymbiotic asso- isms 1–2 billion years ago [1]. These endosymbiotic partner- ciations in which chemosynthetic (sulfur- or methane- ships effectively created ‘new’ organisms that were capable oxidizing) bacteria live directly within the cells of a of invading novel metabolic and ecological niches. Similarly, eukaryotic host. We focus on endosymbioses found at contemporary associations involving intracellular auto- deep-sea hydrothermal vents and present a conceptual trophic Proteobacteria and invertebrates are ubiquitous in model of thioautotrophic (sulfur-oxidizing) metabolism for marine-reducing environments and are particularly well- the vestimentiferan tubeworm Riftia pachyptila, the best characterized in the unique ecosystems structured around studied of all chemosynthetic symbioses (see Ref. [8]). A deep-sea hydrothermal vents [2]. detailed discussion of episymbiotic associations, which Unlike all other major ecosystems on Earth, which are involve bacteria living on the exterior of the host and driven by photosynthesis, hydrothermal vent ecosystems which constitute an important component of the microbial rely on chemosynthesis, a process by which prokaryotic community in reducing environments, is beyond the scope organisms synthesize C3 compounds (e.g. phosphoglycerate) of this review. However, we refer to these associations to from C1 compounds (e.g. CO ,CH ) using chemical energy. 2 4 present important contrasts to endosymbioses (for In this review, ‘chemosynthetic’ describes microorganisms detailed information about episymbioses, see reviews by that: (i) oxidize reduced inorganic compounds (e.g. sulfide) Polz et al. [9] and Ott et al. [10,11]). for energy and fix CO2 for biomass synthesis (i.e. chemo- autotrophy), or (ii) use single carbon compounds as both an energy and carbon source (e.g. methanotrophy) [2–4].At Chemosynthetic symbioses Corresponding author: Cavanaugh, C.M. ([email protected]). The diversity of symbiont-bearing hosts is striking – Available online 28 July 2005 chemosynthetic bacteria are known to associate with www.sciencedirect.com 0966-842X/$ - see front matter Q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.tim.2005.07.007 440 Review TRENDS in Microbiology Vol.13 No.9 September 2005 – – SEAWATER HS O2 NO3 – – Hb-O2-HS Hb-O2-HS – NO3 HS– SO 2– (i) 3 O AMP 2 S (ii) 2e– (iv) ATP PPi SO 2– SO 2– APS H O 4 4 (iii) 2 (v) – – NO3 HCO3 ATP NADPH H+ – CO NO3 2 ADP NADP+ (vi) Calvin (xi) CO2 CO2 benson cycle – NO2 (viii) Organic C, (xii) Translocation symbiont NH3 NH3 biomass SYMBIONT (viii) (ix) Digestion NH3 Translocation (xii) (vii) Organic C, host CO biomass (x) 2 O2 H2O BACTERIOCYTE – HOST BLOOD Hb-O2-HS CO2 Figure 1. Proposed model of metabolism in the symbiosis between Riftia pachyptila and a chemosynthetic sulfur-oxidizing bacterium. Reduced sulfur (primarily HSK) and K K NO3 enter the tubeworm blood from the environment through unidentified transport mechanisms. CO2 and O2 enter by diffusion. In the blood, HS and O2 simultaneously K K and reversibly bind hemoglobin (Hb-O2-HS ) for transport to the trophosome, where these substrates are used in symbiont sulfide oxidation (dashed box). HS is oxidized 2K 2K 2K first to elemental sulfur (S8) or directly to sulfite (SO3 ) (i).SO3 oxidation to sulfate (SO4 ) then proceeds through the APS pathway via the enzymes APS reductase (ii) and ATP sulfurylase (iii), yielding one ATP by substrate level phosphorylation. Electrons liberated during sulfur oxidation pass through an electron transport system, driving oxygen consumption (iv) and the production of ATP and NADPH (v). Fixation of CO2 occurs primarily via ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) in the Calvin Benson cycle (vi), using ATP and NADPH generated from sulfur oxidation. Anapleurotic pathways in both host and symbiont (vii) fix lesser amounts of CO2. Transfer of organic matter from symbionts to host occurs via both translocation of simple nutritive compounds (e.g. amino acids) released by the bacteria (viii) and direct digestion of K symbiont cells (ix). Host oxygen consumption (x) occurs in typical catabolic and anabolic pathways. Nitrate (NO3 ), the dominant nitrogen source for the symbiosis, enters via K K an undescribed transport mechanism and is reduced to nitrite (NO2 ) by the symbionts via an assimilatory nitrate reductase (xi).NO2 is reduced via an uncharacterized 0 pathway to yield ammonia (NH3), which is used for biosynthesis by both symbiont and host (xii). Abbreviations: APS, adenosine 5 -phosphosulfate. Figure modified, with permission, from Ref. [39]. invertebrates from six metazoan phyla, as well as with snail. Snail endobiotic bacteria, in contrast to all charac- ciliate protists (Table 1). These organisms host chemosyn- terized chemosynthetic endosymbionts, reside within cells thetic bacteria either episymbiotically on the exterior of of the host esophageal gland [13]. Although additional the host (e.g. alvinellid tubeworms and rimicarid shrimp) molecular and enzymatic analyses are necessary to or endosymbiotically within cytoplasmic vacuoles of determine the metabolism of these endobacteria, the specialized host cells called bacteriocytes (e.g. vestimenti- reduced digestive system of the snail suggests a reliance feran tubeworms, solemyid clams; see Figure 2). Interest- on symbiont chemosynthesis for host nutrition. This level ingly, two metabolically diverse endosymbionts can co- of adaptation occurs in most other endosymbiont-bearing occur in some invertebrate hosts, such as certain species of invertebrates, in which a reduction or absence of a gut Bathymodiolus mussel, which support both chemoauto- coincides with a complete, or nearly complete, dependence trophs and methanotrophs [12]. Recent work also suggests on internal symbionts for food. Similarly, chemosynthetic that at least one organism, the ‘scaly snail’ found at vents endosymbionts seem to be highly adapted to life in on the Central Indian Ridge, associates simultaneously symbiosis because all attempts to culture these bacteria with both epibiotic and endobiotic bacteria [13]. Epibiotic apart from the host have failed. The inability to culture bacteria of this snail form a dense layer over unique symbionts is undoubtedly due, in part, to our lack of mineralized (iron sulfide) plates covering the foot of the understanding of the bacteriocyte microenvironment.