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REVIEW ARTICLE published: 05 July 2011 doi: 10.3389/fmicb.2011.00146 Close interspecies interactions between from sulfureous environments

Johannes Müller† and Jörg Overmann†∗

Bereich Mikrobiologie, Department Biologie I, Ludwig-Maximilians-Universität München, Planegg-Martinsried, Germany

Edited by: Green are obligate photolithoautotrophs that require highly reducing con- Thomas E. Hanson, University of ditions for growth and can utilize only a very limited number of substrates. These Delaware, USA bacteria thus inhabit a very narrow ecologic niche. However, several green sulfur bac- Reviewed by: Jennifer Macalady, Pennsylvania teria have overcome the limits of immobility by entering into a with motile State University, USA in a type of multicellular association termed phototrophic consortia. James T. Hollibaugh, University of One of these consortia, “Chlorochromatium aggregatum,” has recently been established Georgia, USA as the first culturable model system to elucidate the molecular basis of this symbiotic *Correspondence: interaction. It consists of 12–20 epibionts surrounding a central, Jörg Overmann, Leibniz-Institut, Deutsche Sammlung von chemoheterotrophic betaproteobacterium in a highly ordered fashion. Recent genomic, Mikroorganismen und Zellkulturen, transcriptomic, and proteomic studies of “C. aggregatum” and its epibiont provide insights Inhoffenstraße 7B, 38124 into the molecular basis and the origin of the stable association between the two very Braunschweig, Germany. distantly related bacteria. While numerous of central metabolic pathways are upreg- e-mail: [email protected] †Present address: ulated during the specific symbiosis and hence involved in the interaction, only a limited Johannes Müller and Jörg Overmann, number of unique putative symbiosis genes have been detected in the epibiont. Green Leibniz-Institut, Deutsche Sammlung sulfur bacteria therefore are preadapted to a symbiotic lifestyle. The metabolic coupling von Mikroorganismen und Zellkulturen Braunschweig and between the bacterial partners appears to involve amino and highly specific ultra- Technical University of Braunschweig, structures at the contact sites between the cells. Similarly, the interaction in the equally Inhoffenstraße 7B, 38124 well studied archaeal consortia consisting of and its Ignicoc- Braunschweig, Germany. cus hospitalis is based on the transfer of amino acids while lacking the highly specialized contact sites observed in phototrophic consortia.

Keywords: Chlorochromatium aggregatum, phototrophic consortia, symbiosis, green sulfur bacteria, hospitalis, Nanoarchaeum equitans

INTRODUCTION substrates, they profit energetically from the rapid consumption In their , planktonic bacteria reach total of their excretion products. This renders their ener- numbers of 106 ml–1, whereas in and , 109 and 1011 getically more favorable or makes some reactions even possible bacterial cells·cm–3, respectively, have been observed (Fægri et al., (Bryant, 1979; Zehnder et al., 1982; McInerney, 1986; Schink, 1977; Whitman et al., 1998). Assuming a homogenous distribu- 1992). Recent studies of syntrophic communities in Lake Con- tion,distances between bacterial cells in these environments would stance profundal sediments yielded new and unexpected results. amount to 112 μm for planktonic, 10 μm for envi- The dominant sugar-degrading bacteria were not the typical fer- ronments and about 1 μm for bacteria (Overmann, 2001b). menting bacteria that dominate in anaerobic sludge systems or Taking into account the estimated number of bacterial the environment. They rather represented syntrophic bac- in soil that range from 500,000 (Dykhuizen, 1998)to8.3×106 teria most closely related to the that could only be (Gans et al., 2005), the closest neighbors of each cell statistically grown anaerobically and in coculture with the -using should represent different species. A spatially close association of hungatei (Müller et al., 2008). different bacterial species can result in metabolic complementa- For efficient syntrophic substrate oxidation, close physical con- tion or other synergisms. In this context, the most extensively tact of the partner is indispensable. Monocultures of studied example is the conversion of to and thermopropionicum strain SI and Methanother- in anoxic . The degradation is only pos- mobacter thermautotrophicus show dispersed growth of the cells. sible by a close cooperation of at least four different groups of In contrast, cocultures of the two strains formed tight aggregates bacteria that encompass primary and secondary fermenting bac- when grown on propionate, for which the allowed distance for teria as well as two types of . Along this anaerobic syntrophic propionate oxidation was estimated to be approxi- , end products of one group are exploited by the mem- mately 2 μm(Ishii et al., 2005). Interestingly, the H2-consuming bers downstream the flow of electrons. Although the bacteria partner in syntrophic relationships can be replaced by an H2- involved in the first steps of cellulose degradation do not obligately purging culture vessel, allowing Syntrophothermus lipocalidus to depend on the accompanying bacteria for provision of growth grow on butyrate and Aminobacterium colombiense on alanine in

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pure culture (Adams et al.,2006). Thus,the syntrophic associations (Glaeser and Overmann,2003b).Within the Betaproteobacteria the investigated to date are typically based on efficient H2-removal as central bacterium represents a so far isolated phylogenetic obligate basis for their interdependence. Additional types of bac- belonging to the of the Comamonadaceae. The closest rel- terial interactions have been described more recently. Cultures of atives are Rhodoferax spp., Polaromonas vacuolata and Variovorax were shown to only grow on if paradoxus (Kanzler et al., 2005). in coculture with a chitin degrading bacterium like Aeromonas Based solely on their morphology, 10 different phototrophic hydrophila. In addition to simply growing on the degradation consortia can be distinguished to date (Overmann, 2001a; Over- products produced by the exoenzymes of the partner, P. aerugi- mann and Schubert, 2002). The majority of the morphotypes are nosa induced release of in A. hydrophila by inhibiting its motile, being conferred by the central colorless bacterium. aconitase employing pyocyanin. The resulting incomplete oxida- The 13–69 epibiont cells are either green or brown-colored repre- tion of chitin to acetate by A. hydrophila is then exploited by P. sentatives of the green sulfur bacteria. The smaller consortia like aeruginosa foritsowngrowth(Jagmann et al., 2010). “Chlorochromatium aggregatum” (harboring green epibionts) and Although these well-characterized associations certainly are of “Pelochromatium roseum” (brown epibionts), are barrel shaped major ecological relevance in their respective environments, they and consist of 12–20 epibiont cells (Overmann et al., 1998). Rather were typically obtained using standard defined growth media. globular in shape and consisting of ≥40 epibionts are the sig- As a result, presently available laboratory model systems were nificantly larger consortia “Chlorochromatium magnum” (green selected based on their ability to grow readily and – under at epibionts; Fröstl and Overmann, 2000), “Pelochromatium latum” least some experimental conditions – on their own in pure cul- (brown epibionts; Glaeser and Overmann, 2004) and “Pelochro- ture. Obviously, this cultivation strategy counterselects against matium roseo-viride” (Gorlenko and Kusnezow, 1972). The latter bacterial associations of obligately or at least tight interdepen- consortium is the only one harboring two types of epibionts, dence. While the significant advances in the development of with brown cells forming an inner layer and green ones an outer cultivation-independent techniques permit a partial analysis of layer.“Chloroplana vacuolata” and“Cylindrogloea bactifera”can be so-far-uncultured associations (Orphan et al., 2001; Blumenberg distinguished from the other consortia by their immotility and dif- et al., 2004; Pernthaler et al., 2008), laboratory grown model sys- ferent cell arrangement. “Chloroplana vacuolata” consists of rows tems are still indispensable for in-depth studies of expression of green sulfur bacteria alternating with colorless bacteria forming and metabolism. One model system of prokaryotic associations a flat sheath (Dubinina and Kuznetsov, 1976), with both species that meanwhile can be grown indefinitely in laboratory culture containing gas vacuoles. In “Cylindrogloea bactifera,” a is the phototrophic consortium “Chlorochromatium aggregatum.” containing green sulfur bacteria is surrounding filamentous, col- This consortium represents the most highly developed bacteria– orless bacteria (Perfiliev, 1914; Skuja, 1956). Since they consist of bacteria symbiosis known to date. In parallel, the –archaea two different types of bacteria, the names of consortia are with- association between Ignicoccus hospitalis and Nanoarchaeum equi- out standing in nomenclature (Trüper and Pfennig, 1971) and, tans has emerged as a second laboratory model over the past years accordingly, are given here in quotation marks. (Huber et al., 2003). A comparison between the two model systems When 16S rRNA gene sequences of green sulfur bacteria from that represent two different domains of provides first insights phototrophic consortia were investigated from a total of 14 dif- into the general principles of tight interactions in the prokaryotic ferent lakes in Europe and North America (Glaeser and Over- world. mann, 2004), a total of 19 different types of epibionts could be detected. Of those, only two types occurred on both, the Euro- CHARACTERIZATION OF PHOTOTROPHIC CONSORTIA AND pean and North American continents. Although morphologically ESTABLISHING “CHLOROCHROMATIUM AGGREGATUM”AS identical consortia from one lake always contained just a single A MODEL SYSTEM FOR CLOSE BACTERIAL INTERACTIONS epibiont phylotype, morphologically indistinguishable consortia Phototrophic consortia were already discovered in 1906 (Lauter- from different lakes frequently harbored phylogenetically different born, 1906) and invariably encompass green or brown-colored epibionts. Phylogenetic analyses demonstrated that the epibiont bacteria as epibionts that surround a central bacterium in a highly sequences do not constitute a monophyletic group within the radi- ordered fashion. Several decades later, electron microscopic analy- ation of green sulfur bacteria. Therefore, it was concluded that the ses documented the presence of in the epibiont cells ability to symbiotic interactions was gained independently and led to the conclusion that the phototrophic epibionts belong by different ancestors of epibionts or, alternatively, was present in to the green sulfur bacteria (Caldwell and Tiedje, 1975). This was the common ancestor of the green sulfur bacteria. In parallel, the confirmed by the application of fluorescence in situ hybridiza- phylogeny of central bacteria of phototrophic consortia was inves- tion employing a highly specific oligonucleotide probe against tigated. This analysis exploited a rare tandem rrn arrange- green sulfur bacterial 16S rRNA (Tuschak et al., 1999). The other ment in these bacteria that involves an unusual short interoperon partner bacterium of the symbiosis remained much less investi- spacer of 195 bp (Pfannes et al., 2007). Betaproteobacteria with this gated than its epibionts. First, it had even been overlooked due to genomic feature were exclusively encountered in chemocline envi- its low contrast in the light microscope. Over 90 years later, the ronments and form a novel, distinct and highly diverse subcluster central bacterium was identified as a Betaproteobacterium (Fröstl within the . Within this cluster, the sequences of central and Overmann, 2000) that exhibits a rod-shaped morphology bacteria of phototrophic consortia were found to be polyphyletic. with tapered ends (Overmann et al., 1998). Electron microscopy Thus, like in their green sulfur bacterial counterparts, the ability revealed the cells to be monopolarly monotrichously flagellated to become a central bacterium may have evolved independently in

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several lineages of betaproteobacteria, or was already present in a One feature of green sulfur bacteria which provides interac- common ancestor of the different central bacteria. In the barrel- tion with other prokaryotes is their carbon metabolism. Green shaped types of consortia, the green sulfur bacterial epibionts have sulfur bacteria autotrophically assimilate CO2 through the reduc- overcome their immotility. However, the existence of two differ- tive tricarboxylic cycle. One instantaneous product of pho- ent types of non-motile consortia indicates that motility is not tosynthetic fixation of CO2 is 2-oxoglutarate, and 2-oxo acids the only advantage gained by green sulfur bacteria that form these represent typical excretion products of photosynthesizing cells interspecies association with heterotrophic bacteria. As described (Sirevag and Ormerod, 1970). In natural environments, Chloro- below (see Evidence for Metabolic Coupling), the exchange of bium limicola excretes photosynthetically fixed carbon (Czeczuga metabolites seems to play a major role in the symbiotic interac- and Gradzki, 1973) and thus constitutes a potential electron donor tion, and might therefore be the key selective factor of symbiosis for associated bacteria. Excretion of organic carbon compounds in immotile phototrophic consortia. has also been demonstrated for C. chlorochromatii strain CaD, At present, “Chlorochromatium aggregatum” is the only pho- the epibiont of the phototrophic consortium “Chlorochromatium totrophic consortium that can be successfully cultivated in the aggregatum”(Pfannes, 2007). Vice versa, green sulfur bacteria can laboratory (Fröstl and Overmann, 1998). From the stable enrich- also take advantage of organic carbon compounds produced by ment culture it was possible to isolate the epibiont of the consor- other, for example, fermenting, bacteria. During phototrophic tium in pure culture using deep agar dilution supplemented growth, they are capable of assimilating pyruvate as well as acetate with optimized growth media (Vogl et al., 2006). On the basis of and propionate through reductive carboxylation in the presence 16S rRNA sequence comparisons, the strain is distantly related of CO2 (pyruvate: oxidoreductase; Uyeda and Rabi- ≤ −− to other known green sulfur bacteria ( 94.6% sequence homol- nowitz, 1971)orHCO3 (phosphoenolpyruvate carboxylase; ogy) and therefore represents a novel species within the genus Chollet et al., 1996). The assimilation of organic carbon com- , Chlorobium chlorochromatii strain CaD. However, pounds reduces the amount of electrons required per unit cellular physiological and molecular analyses of the novel isolate did not carbon synthesized. This capability thus enhances photosynthetic reveal any major differences to already described strains of the phy- growth yield and results in a competitive advantage for green sulfur lum Chlorobi. Thus, C. chlorochromatii CaD is obligately anaerobic bacteria. and photolithoautotrophic, and photoassimilates acetate and pep- In their natural environment, green sulfur bacteria are lim- tone in the presence of sulfide and hydrogen carbonate (Vogl et al., ited to habitats where light reaches anoxic bottom waters such as 2006). As a difference to its free-living counterparts the epibiont in thermally stratified or meromictic lakes. Here, cells encounter contains only a low cellular concentration of carotenoids and can- conditions favorable for growth exclusively in a rather nar- not synthesize chlorobactene. A similar anomaly had also been row (typically cm to dm thick) zone of overlap between light observed in the brown epibionts of the phototrophic consortium and sulfide. Compared to other , green sulfur bac- “Pelochromatium roseum” that do not seem to form isorenieratene teria are extremely low-light adapted and capable of exploit- (Glaeser et al., 2002; Glaeser and Overmann, 2003a). In contrast ing minute light quantum fluxes by their extraordinarily large to the epibiont of “Chlorochromatium aggregatum,” all efforts to photosynthetic antenna complexes, the chlorosomes. In contrast cultivate the central bacterium in the absence of its epibionts have to other photosynthetic antenna complexes, the bacteriochloro- failed so far. phyll c, d, or e molecules in chlorosomes are not attached to a scaffold but rather form paracrystalline, tight aggre- PREADAPTATION OF GREEN SULFUR BACTERIA TO gates (Griebenow and Holzwarth, 1989; Blankenship et al., 1995). SYMBIOSIS Until recently, the heterogeneity of pigments complicated the Green sulfur bacteria (Family Chlorobiaceae) constitute a phy- identification of the structural composition of chlorosomes. logenetically distinct lineage within the Chlorobi of the When a Chlorobaculum tepidum triple mutant that almost exclu- Bacteria (Overmann, 2001a). Recently, the chemotrophic sively harbors BChl d was constructed, a syn-anti stacking of Ignavibacterium album gen. nov. sp. nov.,was described (Iino et al., monomers and self-assembly of into tubu- 2010), this novel isolate represents a deeply branching phyloge- lar elements could be demonstrated within the chlorosomes netic lineage and hence a new within the phylum Chlorobi, (Ganapathy et al., 2009). whereas all green sulfur bacteria sensu stricto that are known to date Since they minimize the energetically costly protein synthesis, represent strictly anaerobic photolithoautotrophs. Since a consid- chlorosomes represent the most effective light harvesting system erable number of different 16S rRNA gene sequence types of green known. Up to 215,000 ± 80,000 molecules (in sulfur bacteria engaged in a symbiotic association with the central Chlorobaculum tepidum; Montano et al., 2003) can constitute a Betaproteobacteria (see Characterization of Phototrophic single , that is anchored to 5–10 reaction centers in Consortia and Establishing “Chlorochromatium aggregatum”asa the cytoplasmic membrane (Amesz, 1991). This ratio of chloro- Model System for Close Bacterial Interactions), green sulfur bac- phyll to reaction center is orders of magnitudes higher compared teria may be specifically preadapted to symbiosis and the advent of with other photosynthetic antenna structures. In the phycobili- symbiotic green sulfur bacterial epibionts during may somes of the ratio is 220:1 (Clement-Metral et al., have involved only limited genomic changes. Indeed, several of the 1985), 100–140:1 in light harvesting complex II of anoxygenic physiological characteristics of green sulfur bacteria are regarded phototrophic (Van Grondelle et al., 1983) and 28:1 as preadaptive traits for interactions with other prokaryotes. in the light harvesting complex I (Melkozernov et al., 2006). The

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enormous size of the photosynthetic antenna of green sulfur bac- the central bacterium. This question was addressed by adding teria enables them to colonize extreme low-light habitats up to carboxyfluorescein diacetate succinimidyl (CFDA-SE) to depths of 100 m in the Black (Overmann et al., 1992; Marschall intact consortia and following fluorescence in epibionts and cen- et al., 2010) or below layers of other phototrophic organisms like tral bacteria over (Bayer, 2007). CFDA-SE enters cells by sulfur bacteria (Pfennig, 1978). Commensurate with their diffusion and, after cleavage by intracellular esterase , adaptation to extreme light limitation, green sulfur bacteria also confers fluorescence to the bacterial cell. Central bacteria were exhibit a significantly reduced maintenance requirement already detectable after 2 min of exposure (Figure 1A), whereas compared to other bacteria (Veldhuis and van Gemerden, 1986; epibionts in the same sample could only be detected after 12 min Overmann et al., 1992). Chlorobium phylotype BS-1 isolated from of incubation with CFDA-SE and developed only weak fluores- the Black Sea maintained a constant level of cellular ATP over cence (Figure 2). The fluorescence activity of the central rod 52 days, if exposed to low-light intensities of 0.01 mmol quanta remained strong throughout the experiment which is indicative m–2 s–1 (Marschall et al., 2010). The high efficiency of green sulfur of a higher esterase activity than in the epibiont. Since the cen- bacteria allows them to colonize habitats in which other photosyn- tral bacterium is presumably heterotrophic, it is likely to express thetic bacteria are unable to grow. A chemotrophic bacterium that esterases such as lipases at a higher number or intracellular con- associates with green sulfur bacteria and is capable of exploiting centration. These results indicate that, even in intact phototrophic part of their fixed carbon thus would gain a selective advantage consortia, diffusion of small water-soluble molecules toward the during evolution. central bacterium is not significantly impeded by the surrounding layer of epibionts. Therefore, sensing of sulfide by the central bac- SELECTIVE ADVANTAGE OF CONSORTIA FORMATION terium itself, eliciting the sulfide chemotactic response observed Free-living representatives of green sulfur bacteria are immotile for the consortia is feasible. However, in analogy to the proposal and only species able to produce gas vacuoles can regulate their that heterotrophic bacteria colonizing the heterocysts of cyanobac- vertical position. However, changes in buoyant density mediated teria may shield them from high ambient O2 concentrations (Paerl by gas vesicle production occur only over time periods of several and Kellar, 1978), one could speculate that the consumption of days (Overmann et al., 1991). Due to the motility that is conferred sulfide by the epibionts might decrease the concentration of sul- by the flagellated central bacterium, the consortium can orien- fide reaching the central bacterium. Thus, if sensing of sulfide is tate itself much faster in light and sulfide gradients and reaches carried out by the central bacterium, the photosynthetic activity locations with optimal conditions for in a shorter of the epibiont could have a regulatory function regarding the period of time. In fact, “C. aggregatum” has been found to vary chemotaxis of the consortium toward sulfide. its position rapidly across the chemocline in two Tasmanian lakes The orientation of the consortium toward light and sul- (Croome and Tyler, 1984). A scotophobic response, that is swim- fide is of special interest since probably neither of these ming away from darkness toward light, has been demonstrated attractants is used in the metabolism of the motile central for intact consortia in the laboratory (Fröstl and Overmann, 1998; Glaeser and Overmann, 2003a) and leads to a rapid accumulation of consortia in (dim)light. In addition, laboratory cultures as well as natural populations of phototrophic consortia exhibit a strong chemotaxis toward sulfide (Fröstl and Overmann, 1998; Glaeser and Overmann, 2003b). In contrast to light, the spatial distribution of sulfide does not necessarily occur in a strictly vertical gradient in the natural habi- tat. In analogy to the presence of point sources of organic carbon substrates (Azam and Malfatti, 2007) that attract chemotrophic aquatic bacteria in zones extending tens to hundred microme- ter around the sources (Krembs et al., 1998), organic sinking into anoxic water layers may develop into hot spots of sulfate reduction. Due to the motility of phototrophic consor- tia, the otherwise immotile green sulfur bacteria epibionts would gain rapid access and thus a highly competitive advantage over their free-living relatives especially in laterally inhomogeneous environments. If photosynthetically fixed carbon is indeed trans- ferred to the central bacterium, however, the net balance of the increased availability of sulfide and the loss of electrons to the central bacterial partner must still be positive for the green sulfur bacterium. The tight packing of epibiont cells in the phototrophic con- FIGURE 1 | CFDA-SE stained phototrophic consortia. CR, central rod; sortium raises the question whether dissolved compounds can EB, epibiont. (A,B)2(A) and 5 min (B) of ; only the central bacterium is visible. (C) 12 min of staining; epibionts become visible. (D) actually diffuse into the consortium and reach the central bac- 15-min staining; whole consortium stained. terium or if the epibionts represent a diffusion barrier around

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syntrophic cocultures of Chlorobium phaeovibrioides and Desul- furomonas acetoxidans. In these cocultures, acetate is oxidized by Desulfuromonas acetoxidans with sulfur as , which leads to a recycling of the sulfide that can then be used again for anoxygenic photosynthesis by Chl. phaeovibrioides. Only minute amounts of sulfide (10 μM) are required to keep this running (Warthmann et al., 1992). Similarly, sulfate reduc- ers are able to grow syntrophically with green sulfur bacteria with only low equilibrium concentrations of sulfide (Biebl and Pfennig, 1978). In addition, such interactions may also encompass trans- fer of organic carbon compounds between the partners. In mixed cultures of Desulfovibrio desulfuricans or D. gigas with Chloro- bium limicola strain 9330, ethanol is oxidized to acetate with sulfate as electron acceptor and the acetate formed is incorpo- rated by Chl. limicola such that ethanol is completely converted to cell material. However, the hypothesis of a sulfur cycling within pho- FIGURE 2 |Time course of fluorescence intensities of CFDA-SE stained totrophic consortia became less likely by the discovery that the central bacteria and epibiont cells. central bacterium belongs to the Betaproteobacteria, whereas only the or encompass typi- cal sulfur- or sulfate-reducers (Fröstl and Overmann, 2000). bacterium. From the perspective of the epibiont, relying on a As shown above, the exchange of sulfur compounds has non-photosynthetic partner for transportation would pose the been established across a physiologically and phylogenetically risk of being carried into dark, and/or sulfide-free unfavor- diverse range of prokaryotes. But those symbiotic interactions able deeper water layers. Consortia formation thus would either were not accompanied by consortia formation. It is thereby require the expression of a suitable chemotactic response (i.e., concluded, that sulfur cycling does not appear to be suffi- toward sulfide) and of a suitable photosensor in the central bac- ciently selective to explain the advent of phototrophic consortia terium or effective means of interspecific communication. Acqui- during evolution. sition of these traits must have been a critical stage and hap- pened early during the coevolution of the bacterial partners in MOLECULAR BASIS OF THE SYMBIOTIC INTERACTION IN phototrophic consortia. PHOTOTROPHIC CONSORTIA In recent ultrastructural studies of the central bacterium of FEATURES OF THE EPIBIONT THAT RELATE TO SYMBIOSIS “C. aggregatum,” conspicuous 35-nm-thick and up to 1-μm-long In to make a first assessment of the imprint of symbiotic zipper-like crystalline structures were found that resemble the lifestyle on the genome of the epibiont, the genome features of C. chemotaxis receptor Tsr of (Wanner et al., 2008). chlorochromatii CaD can be compared to those of Nanoarchaeum In a comparative ultrastructural study of 13 distantly related equitans and Ignicoccus hospitalis in the archaeal consortia. organisms harboring chemoreceptor arrays from all seven major Nanoarchaeum equitans is the representative of a new archaeal signaling domain classes, receptors were found to possess an phylum and most likely represents a parasitic universal structure which has presumably been conserved over epibiont of Ignicoccus.TheN. equitans genome comprises only long evolutionary distances (Briegel et al., 2009). The prominent 491 kb and encodes 552 genes, rendering it the smallest genome ultrastructure discovered in the central bacterium exhibits sev- for an exosymbiont known to date (Waters et al., 2003). Genome eral similarities to the chemoreceptors reported and provides a reduction includes almost all genes required for the de novo first indication that a chemotaxis receptor is present in the central biosynthesis of amino acids, , cofactors, and as bacterium. well as many known pathways for carbon assimilation. Commen- One characteristic feature of green sulfur bacteria that pro- surate with this findings, the Nanoarchaeum equitans epibiont vides a basis for interaction with other bacteria is the extracellular appears to acquire its lipids from its host (Waters et al., 2003). Yet, deposition of sulfur globules (zero valence sulfur), the initial prod- the N. equitans genome harbors only few pseudogenes or regions uct of sulfide oxidation during anoxygenic photosynthesis. This of non-coding DNA compared with of obligate bacterial sulfur is further oxidized to sulfate only after depletion of sul- symbionts that undergo reductive evolution, and thus is genom- fide. The extracellular deposition renders the sulfur available to ically significantly more stable than other obligate parasites. This other bacteria such as, for example, sulfur reducers. Therefore, it has been interpreted as evidence for a very ancient relationship had initially been proposed that the central bacterium of pho- between N. equitans and Ignicoccus (Brochier et al., 2005). The totrophic consortia is a sulfate- or sulfur-reducing bacterium. recent bioinformatic analysis also suggest that N. equitans repre- In that case, extracellular sulfur produced by the green sulfur sents a derived, fast-evolving euryarchaeal lineage rather than the bacteria could be utilized by the central bacterium to establish representative of a deep-branching basal phylum (Brochier et al., a close sulfur cycle within the phototrophic consortium (Pfen- 2005). With a size of 1.30 Mbp and 1494 predicted open reading nig, 1980). Such a sulfur cycle has been established in defined frames (ORFs), the genome of I. hospitalis shows a pronounced

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genome reduction that has been attributed to the reduced meta- provides a large number of putative symbiosis genes, the num- bolic complexity of its anaerobic and autotrophic lifestyle and an bers are rather low compared to the unique and unknown ORFs highly efficient adaptation to the low energy yield of its metabolism in the other green sulfur bacteria (Table 1). Even if it is assumed (Podar et al., 2008). that all of these unknown genes encode for involved Similar to I. hospitalis, the genome size of Pelagibacter ubique in symbiosis, this number is still rather small compared to the (1.31 Mbp and 1354 ORFs) so far marks the lower limit of free- 1387 genes encoding niche-specific functions in enterohemor- living organisms. It exhibits hallmarks of genome streamlining rhagic E. coli O157:H7 (Perna et al., 2001). Low numbers of such as the lack of pseudogenes, , transposons, extrachro- niche-specific genes have been reported for enterica mosomal elements, only few paralogs, and the shortest intergenic or and have been interpreted as indication for spacers yet observed for any cell (Giovannoni et al., 2005). This preadaptation of the non-pathogenic ancestor. This supports the likely reduces the costs of cellular replication (Mira et al., 2001). hypothesis of a preadaptation of green sulfur bacteria to sym- As a second feature, the genome of P. ubique has a G:C content biosis. From a broader perspective, the discovery of putative of 29.7% that may decrease its cellular requirements for fixed symbiosis genes in the epibiont genome that resemble typical (Dufresne et al., 2005). bacterial virulence factors suggest that modules thought to be By comparison, a much larger genome size of 2.57 Mbp has limited to bacterial are employed in a much wider been determined for C. chlorochromatii CaD. This size represents biological context. the average value of the 11 other publicly available genomes of green sulfur bacteria (Table 1). Thus, a reduction in genome THE REGULATORY RESPONSE EVOKED BY SYMBIOSIS INVOLVES size that is characteristic for bacterial (Anders- GENES OF THE NITROGEN METABOLISM son and Kurland, 1998; Moran et al., 2002, 2008) and for the When the of C. chlorochromatii CaD in the free- archaeal consortium (Podar et al., 2008) did not occur during living state was compared to that of the symbiotic state by 2-D the evolution of the epibiont of “C. aggregatum.” This suggests differential gel electrophoresis (2-D DIGE), it became appar- (i) a shorter period of coevolution of the two partner bacteria in ent that symbiosis-specific regulation involves genes of central phototrophic consortia, (ii) a significantly slower rate of evolu- metabolic pathways rather than symbiosis-specific genes (Wen- tion of their genomes, or (iii) that the genome of C. chlorochro- ter et al., 2010). In the soluble proteome, 54 proteins were matii is not undergoing a streamlining process as observed in expressed exclusively in consortia. Among them were a consid- other symbiotic associations. The latter suggestion would indi- erable number of proteins involved in metabolism. cate a lack of selective advantage for “Chl. aggregatum”from These included , 2-isopropylmalate synthase, genome streamlining. and the nitrogen regulatory protein P-II. The latter showed Wet-lab and in silico analyses of the epibiont genome revealed the highest overall upregulation that amounted to a 189-fold the presence of several putative symbiosis genes. An initial com- increase in transcript as determined by subsequent bination of suppression subtractive hybridization with bioinfor- RT-qPCR. It is thereby concluded that the amino acid require- matics approaches identified four ORFs as candidates. Two of ment in the consortium is higher than in the epibiont in the ORFs (Cag0614 and 0616) exhibit similarities to putative pure culture. filamentous hemagglutinins that harbor RGD (arginine–glycine– Parallel investigations of the membrane proteome revealed that aspartate) tripeptides. In , hemagglutinins a branched chain amino acid ABC-transporter binding protein with these motifs are involved in the attachment to mammalian was expressed only in the associated state of the epibiont. Inter- cells (Vogl et al., 2008). Most notably, a comparative study of estingly, the expression of the ABC-transporter binding protein 580 sequenced prokaryotic genomes revealed that Cag 0614 and could also be induced in the free-living state by addition of sterile 0616 represent the largest genes detected in prokaryotes so far. filtered supernatant of the consortia culture, but not with peptone In fact, Cag 0616 is only surpassed in length by the exons or branched chain amino acids themselves. This is an evidence for of the human titin gene (Reva and Tümmler, 2008). The two a signal exchange between the two symbiotic partners mediated other genes detected (Cag1919 and 1920) resembles repeats in through the surrounding medium. toxin (RTX)-like proteins and hemolysins, respectively. All four The results of the proteome analysis were supplemented by genes have in common that they are unique in C. chlorochro- transcriptomic studies of the epibiont in the associated and the matii CaD and that certain domains of their inferred products free-living state (Wenter et al., 2010). Of the 328 differentially are only known from bacterial virulence factors. If the four expressed genes, 19 genes were found to be up-regulated and are genes were not misassigned, they are potentially involved in involved in amino acid synthesis while six genes of the amino the symbiotic interaction between the two partner bacteria in acid pathways were down-regulated. The conclusion that nitrogen phototrophic consortia. metabolism of the epibiont is stimulated in the symbiotic state is To identify additional symbiosis genes, in silico subtractive commensurate with the simultaneous up-regulation of the nifH, hybridization between the genome sequence of C. chlorochro- nifE, and nifB genes and with the prominent expression of the P-II matii CaD and the other 11 sequences of green sulfur bac- nitrogen regulatory protein. terial genomes was performed. This yielded 186 ORFs unique The results of the proteome analyses indicates that (i) a signal to the epibiont (Wenter et al., 2010), 99 of which encode for exchange occurs between the central bacterium and the epibiont hypothetical proteins of yet unknown function. Although this that controls the expression of symbiosis relevant genes and (ii)

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Table 1 | Comparison of green sulfur bacterial genomes against each other.

Green sulfur bacterium Genome ORFs-in Unique Genome Unknown G + C size (Mbp) genome ORFs (%) unique ORFs (%)

Chlorobium chlorochromatii CaD3 2.57 2002 186 9.3 99 44.3 Chlorobaculum parvum NCIB 8327 2.29 2078 139 6.7 81 55.8 Chlorobaculum tepidum TLS 2.15 2252 396 17.6 366 56.5 Chlorobium ferrooxidans DSM 13031 2.54 2158 181 8.4 84 50.1 Chlorobium limicola DSM 245 2.76 2522 204 8.1 149 51.3 Chlorobium phaeobacteroides BS1 2.74 2559 331 12.3 211 48.9 Chlorobium phaeobacteroides DSM 266 3.13 2743 266 9.7 164 48.4 Chlorobium phaeovibrioides DSM 265 1.97 1773 56 3.2 33 53.0 Chloroherpeton thalassium ATCC 35110 3.29 2731 971 35.6 396 45.0 Pelodictyon phaeoclathratiforme BU-1 3.02 2911 550 18.9 394 48.1 Chlorobium luteolum DSM 273 2.36 2083 100 4.8 49 57.3 Prosthecochloris aestuarii SK413, DSM 271 2.58 2402 409 17.0 215 50.1 Average 2.61 2351.2 315.8 12.6 186.8 50.1.

In silico subtractive hybridization was conducted with the Phylogenetic Profiler available at the DOE Joint genome Institute website (http://img.jgi.doe.gov). metabolic coupling between C. chlorochromatii and the central only the addition of acetate and peptone stimulated the growth of Betaproteobacterium may involve amino acids. Metabolic coupling the epibiont of “C. aggregatum”(Vogl et al., 2006). It remains has also been detected in the two-membered to be tested whether transfer of organic carbon in this form consisting of Anabaena sp. strain SSM-00 and Rhizobium sp. occurs in the opposite direction from the central bacterium to strain WH2K. Between the two species, nanoscale secondary the epibiont. mass spectrometry (nanoSIMS) analyses indicated an exchange Stable isotope signatures (13C) of I. hospitalis and N. equitans of metabolites containing 13C and 15N fixed by the heterocysts were analyzed to investigate a possible carbon transfer between the of the filamentous cyanobacteria to the attached epibiont cells two archaeal partners. Labeling patterns of Ignicoccus amino acids (Behrens et al., 2008). grown in the coculture as well as in pure culture were compared to those in the Nanoarchaeum amino acids. Therefore, amino acids 13 EVIDENCE FOR METABOLIC COUPLING were separated by chromatography and incorporation of Cat The proteomic and transcriptomic evidence described in the specific carbon positions was identified by NMR spectroscopy. preceding paragraphs point toward an exchange of metabolites The labeling patterns from all three cultures were exactly iden- between the two symbiotic partners of “C. aggregatum.” Mean- tical. In addition, genes involved in the de novo biosynthesis of while, more direct evidence for a transfer of carbon between the amino acids are missing in the Nanoarchaeum genome. Based on bacterial partners of phototrophic consortia has been obtained. this combined evidence, it was concluded that amino acids are In a series of labeling experiments with 14C, a rapid exchange transferred from the I. hospitalis host to the N. equitans cells of labeled carbon from the epibiont to the central bacterium (Jahn et al., 2008). In addition, cellular seem was observed (Johannes Müller and Jörg Overmann, unpublished to be exchanged between the partners in the archaeal consor- observations). External addition of several amino acids as well tia. In the latter, LC–MS analyses of membrane lipids in their as 2-oxoglutarate to the inhibited this carbon intact polar forms showed very similar chemical patterns in both exchange. Together with the observed excretion of photosyntheti- organisms with and caldarchaeol constituting the main cally fixed carbon by C. chlorochromatii CaD, these results suggest core lipids. Furthermore, stable isotope labeling (13C) yielded a transfer of newly synthesized small molecular weight organic nearly identical results for the hydrocarbons derived from N. to the central bacterium. Such a transfer may provide the equitans and I. hospitalis. Those results, combined with a lack central bacterium with a selective advantage in illuminated sul- of genes for biosynthesis in the genome of N. equitans led fidic environments where degradation of organic matter proceeds to the conclusion that lipids in the archaeal consortium are syn- mainly through the anaerobic food chain and involves thesized in I. hospitalis and transported to its partner of chemoheterotrophs for organic carbon compounds. By trans- (Jahn et al., 2004). ferring amino acids, the epibiont may not only support growth of Amino acids also seem to be of central importance for other the central bacterium with respect to carbon, but also to nitrogen symbioses. The deep sea tube is depen- and even sulfur. dent on arginine supplied by its bacterial endosymbionts (Minic Todate it has remained unclear whether the association in pho- and Hervé, 2003), whereas in legume-root nodules, amino acids totrophic consortia also offers an additional advantage for the are used as both an ammonium and a carbon shuttle (Lod- green sulfur bacterial epibiont apart from the gain of motility and wig et al., 2003). Interestingly, Rhizobia become symbiotic aux- the resulting potential increase in sulfide supply. Extensive sub- otrophs for branched chain amino acids after infecting the host strate utilization assays with C. chlorochromatii CaD revealed that due to a downregulation of the respective biosynthetic

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pathways, making them dependent on the supply by the plant because the highly fluorescent dye calcein should have dif- (Prell et al., 2009). fused after its formation from the epibiont cells into the central bacterium. Furthermore, after subsequent bleaching of one of MECHANISMS OF METABOLITE EXCHANGE the epibiont cells (arrowheads in Figures 4B,C) using confocal The very structure of the phototrophic consortium in itself facil- microscopy, a recovery of fluorescence could not be detected in itates the putative transfer of compounds from one partner to the bleached cell, which excludes the possibility of free diffu- the other since the direct cell–cell contact prevents a diffusion sion between the epibiont cells through the interconnecting pili of compounds over larger distance and hence minimizes transfer (Figures 4B,C). time. Theoretically, the metabolic coupling of the two partners of A similar experiment has been conducted with the filamen- the consortium “C. aggregatum” may be based on an unspecific tous cyanobacterium Anabaena cylindrica which is considered to excretion of substrates by the epibiont followed by the uptake via be a truly multicellular . Single calcein stained cells the central bacterium, or involve specific molecular structures and within an Anabaena filament fully recovered calcein fluores- mechanisms of substrate exchange. Several observations indicate cence 12 s after bleaching. This effect was ascribed to intercellular that the latter is the case in “C. aggregatum.” channels allowing free diffusion of molecules from The ultrastructure of the contact sites in “C. aggregatum” to cytoplasm (Mullineaux et al., 2008). Such a rather unspecific and in the archaeal consortia have been studied in detail using transfer is unlikely to occur across the contact site of the pho- different electron microscopy approaches (Junglas et al., 2008; totrophic consortium “C. aggregatum” where it must be much Wanner et al., 2008). In free-living epibionts, as well as in all more substrate-specific. other known green sulfur bacteria, chlorosomes are distributed evenly among the inner face of the cytoplasmic membrane. CONCLUSIONS However, in the associated state, chlorosomes are absent in the While the different types of symbioses and syntrophic associa- green sulfur bacterial epibionts at the site of attachment to the tions discussed in the preceding sections all provide an energetic central bacterium. Replacing the antenna structures, a 17 nm- advantage to one or both partners, only few of these associations thick layered structure of yet unknown function has been dis- reached the level of organizational complexity of the highly struc- covered (Vogl et al., 2006; Wanner et al., 2008). Interestingly, tured, permanent consortia. Thus, a permanent cell–cell contact is treatment of the epibionts with the extracellular cross-linkers not mandatory in the case of syntrophic cultures in which deple- DTSSP and BS3 revealed the branched chain amino acid ABC- tion of substrates can lead to disaggregation of the associations transporter binding protein (compare The Regulatory Response (Peduzzi et al., 2003). The highly developed and obligate inter- Evoked by Symbiosis Involves Genes of the Nitrogen Metabo- action in phototrophic consortia is likely to be related to the lism) to be cross-linking with other proteins, indicating that it pronounced energy limitation in the low-light habitats and to is localized at the cell surface or in the (Wenter et al., the efficient and regulated exchange of metabolites. Phototrophic 2010). In contrast to the epibionts of the phototrophic consortium “Chlorochromatium aggregatum” that maintain a permanent cell– cell contact to the central bacterium, Nanoarchaeum equitans has been observed in different states of attachment to Ignicoccus hos- pitalis. The surface structures of the two organisms may either be in direct contact or, alternatively, in close vicinity to each other. In the latter case, fibers bridging the gap between the cells are clearly visible. In “C. aggregatum,” connections between the two part- ner bacteria stretching out from the central bacterium are more FIGURE3|(A)Scanning electron photomicrographs of a partially disaggregated consortium, E, epibiont, CB, central bacterium, arrows prominent than in the archaeal consortium. Periplasmic tubules pointing toward periplasmic tubules (PT). (B) Transmission electron (PT) are formed by the outer membrane and are in linear contact photomicrographs of ultra-thin sections showing elongated PT at the tip of with the epibionts (Figure 3A). The PT are distributed over the the central bacterium. Modified after Wanner et al. (2008). entire cell surface (Figure 3B) and reach 200 nm in length at the poles of the central bacterium. It had been speculated that the periplasmic tubules repre- sent connections of a shared periplasmic (Wanner et al., 2008). However, this could not be confirmed by fluorescence recovery after photobleaching (FRAP)-analyses (Johannes Müller and Jörg Overmann, unpublished observations). After stain- ing of the consortia with calcein acetoxymethylester (calcein AM), only the epibionts but not the central bacterium could be detected by fluorescence microscopy (Figure 4A). Obvi- FIGURE 4 | “Chlorochromatium aggregatum” consortia after calcein ously, the central bacterium (arrows in Figure 4A) is lacking AM staining. (A) Arrows pointing toward unstained central bacteria. (B,C) Consortium before and after photo-bleaching; arrows pointing toward an esterase specific for cleaving calcein AM. This result in itself bleached epibiont cell. already contradicts the hypothesis of a combined periplasm

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consortia harboring other prokaryotes than green sulfur bacteria help to determine whether and which additional preadaptations have not been found so far, emphasizing the role of preadaptation of the betaproteobacterium were essential for establishing of the green sulfur bacterial partner for the development of the this symbiosis. symbiosis that was augmented by the gain of specific functions such as genes similar to virulence genes, periplasmic tubules ACKNOWLEDGMENTS for cell–cell contact or the intracellular sorting of chlorosomes We thank Anne Bayer for supplying material for Figures 1 and 2 in the epibionts. The gain of motility by the epibiont seems and Andreas Binder, , Department Biology I, University to constitute a selective advantage that led to the coevolution of Munich, for help with confocal laser scanning microscopy. This with a motile betaproteobacterium. The recent completion of work was supported by a grant of the Deutsche Forschungsge- the central bacterial genome sequence for “C. aggregatum” should meinschaft to Jörg Overmann (grant DFG OV20/10-2).

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