PERSPECTIVES

MICROBIOLOGY

Survival of a polychaete worm in a deep A Proteomic Snapshot of sea hydrothermal vent depends on complex metabolic interactions with symbiotic Life at a Vent bacteria. Charles R. Fisher and Peter Girguis

n the late 1970s, scientists discovered rich Hydrothermal vents are dynamic and its symbionts with a bountiful supply of both communities of animals living around potentially dangerous environments. Temp- (4). In return, the symbionts are extremely Ideep-sea hydrothermal vents in the eastern eratures can range from near freezing to more efficient and productive, fixing carbon at high Pacific. Since then, hundreds of other chemo- than 300°C over centimeters. Within animal rates to support the host’s growth (5). This autotrophic-based communities and animals communities, temperatures can vary over suite of adaptations enables Riftia to be very have been discovered. What were once thought more than 40°C within seconds. Hydro- fast growing and quite fecund, while reliant on of as biological oddities are now confirmed to thermal vents are also quite ephemeral, with its symbionts for nutrition. be abundant in all the world’s oceans. What the local sources of hydrothermal flow often last- These and earlier studies of Riftia focused sites all have in common is an abundance of ing only a few years. Consequently, the habitat on characterizing its major biochemical, physi- reduced chemicals (such as sulfide or methane) fluctuates, and vent fauna must balance expo- ological, and ecological attributes, such as at the sea floor. Many of the sites, including sure to the hot and potentially toxic vent fluid hemoglobin properties, oxygen uptake rates, mineral-rich hydrothermal vents and the oil- with the need to obtain nutrition either directly and habitat characteristics. More recent studies and gas-rich hydrocarbon seeps, are increas- from the fluid or from microbes living in the have grown in both breadth and depth, investi- ingly affected by anthropogenic activities. To fluid. Riftia is supremely adapted for its sym- gating the expression of genes, quantifying the assess the effects of these activities, we must biotic life style in this environment. They live metabolic interactions between host and sym- better understand the lifestyles of the crea- with their highly vascularized gill-like plumes biont, and describing the ecological dynamics tures that thrive in this extreme habitat. The exposed to vent fluid and have circulating of Riftia aggregations. Markert et al. have now use of powerful molecular tools, such as the hemoglobins that bind to both oxygen and sul- used the power of genomic analyses coupled approach described by Markert et al. on page fide reversibly and with high affinity and with high-throughput protein profiling to 247 of this issue, will advance our understand- capacity (2, 3). This allows Riftia to take up obtain a snapshot of the proteins (or proteome) ing of the abundant chemoautotrophic sym- and store large amounts of these chemo- expressed by the Riftia symbiont. Their results bioses of the deep sea. autotrophic substrates, transport them through illustrate the degree to which Riftia symbionts One of the most prominent members of the its tissues with no harmful effects, and provide are poised for high rates of chemoautotrophic hydrothermal vent community is the tube- carbon fixation powered by sulfide worm Riftia pachyptila, a siboglinid poly- oxidation. For example, Markert chaete that has become the unofficial poster et al. find that 12% of the total child for hydrothermal vents. Riftia has no cytosolic proteome of these sym- mouth, gut, or anus and cannot feed by normal bionts consists of three proteins means. Instead, Riftia depends on intracellular involved in coupling energy pro- chemoautotrophic symbionts—which fill a duction to sulfide oxidation. This is large internal organ called the trophosome— a marked departure from fast- for nutrition. The symbionts are γ-proteo- growing, free-living heterotrophic bacteria, which are functionally analogous to bacteria that instead expend a con- plant chloroplasts in that they generate siderable fraction of their energy organic carbon as a food source for their worm synthesizing amino acids for their host (using sulfide as an electron donor and own cell division and growth (6). oxygen as an electron acceptor). Key to under- The prominence of these three pro- standing the biology of Riftia and other teins underscores the central role chemoautotrophic symbioses is an under- of the symbionts: to provide nutri- standing of the biology of their symbionts. tion to the association by harness- However, no chemoautotrophic symbiont has ing energy from sulfide. ever been cultured in a laboratory, and this has The mechanism of inorganic long hampered our ability to study their carbon uptake and transport by metabolism. Markert et al. use a proteomic the host, and fixation by the approach to examine protein expression in symbiont, has been the subject of Riftia symbionts and gain new insights into much inquiry and debate. The their biochemistry and metabolism (1). first indication that hydrothermal vent fauna obtain their nutrition C. R. Fisher is in the Department of Biology, Pennsylvania from chemoautotrophic sources State University, University Park, PA 16801, USA. cfisher@ (not from photosynthetically de- psu.edu P. Girguis is in the Department of Organismic and Evolutionary Biology, Harvard University, Cambridge, MA The life aquatic. Riftia pachyptila tubeworms at a hydrothermal rived products but from primary

02138, USA. E-mail: [email protected] vent field located near 21°N on the East Pacific Rise. production powered by hydrogen AQUARIUM RESEARCH INSTITUTE BAY VRIJENHOEK/MONTEREY B. CREDIT:

198 12 JANUARY 2007 VOL 315 SCIENCE www.sciencemag.org

Published by AAAS PERSPECTIVES sulfide) came from analyses of their stable Far less than 1% of the microbes present in transmission of the symbionts between gen- carbon (13C) and nitrogen (15N) isotope con- nature have been successfully cultured in the erations is not direct because the larvae are tents (7, 8). The amount of these isotopes laboratory. No chemoautotrophic symbiont aposymbiotic and newly settled tube worms detected in dominant fauna (tube worms, has yet been cultured, and it is possible that must acquire their symbionts anew each mussels, and clams) did not reflect normal many never will be. Not only is the milieu of a generation from an apparently free-living deep-sea carbon and nitrogen. Later studies living host difficult to imitate in vitro, but in pool. How is the metabolism of the free- demonstrated the presence of intracellular some cases, the exchange and integration of living stage different from that of the symbi- chemoautotrophic symbionts in these ani- host and symbiont genes may have yielded a otic stage? Once contact with a host is made, mals, confirming the nonphotosynthetic symbiont more analogous to an organelle than how do the symbionts contribute to success- nutritional source of carbon and nitrogen. to a free-living microbe. In such instances, ful establishment of the symbiosis? Mole- Mussels and clams had δ3C values of about genomic and proteomic approaches provide cular approaches like that of Markert et al. –30 per mil (‰), in the range that was valuable information on the symbiont’s meta- may help answer such questions about life expected for carbon that is derived from bolic capabilities and evolutionary history. and relationships in this remote and inhos- chemoautotrophic bacteria. However, the Quantitative proteomics has the additional pitable environment. δ13C value of Riftia was much higher (~ value of allowing one to use protein expres- –15‰) and consequently more difficult to sion levels as a metric for studying the impor- References 1. S. Markert et al., Science 315, 247 (2007). understand. A variety of explanations have tance of metabolic pathways used by these 2. A. J. Arp, J. J. Childress, Science 213, 342 (1981). been put forward to explain these isotope symbiotic microbes in situ. 3. A. J. Arp, J. J. Childress, Science 219, 295 (1983). values, but none has proven completely satis- Many questions remain about these enig- 4. J. J. Childress, C. R. Fisher, Oceanogr. Mar. Biol. Annu. Rev. 30, 337 (1992). factory (9–11). matic animals and their rather extreme life 5. P. R. Girguis, J. J. Childress, J. Exp. Biol. 209, 3516 Markert et al. find high amounts of styles. Riftia’s trophosome, which is packed (2006). enzymes involved in the reductive tricar- with billions of bacteria per gram of tissue, 6. H. Akashi, T. Gojobori, Proc. Natl. Acad. Sci. U.S.A. 99, 3695 (2000). boxylic acid cycle in extracts of the Riftia is intertwined with the animal’s gonads. 7. G. H. Rau, J. I. Hedges, Science 203, 648 (1979). symbiont and suggest that this is an important Considering the rarity of active hydrother- 8. G. H. Rau, Can. J. Fish. Aquat. Sci. 37, 742 (1980). pathway of carbon fixation by the symbiont. mal vents on the sea floor, and the improba- 9. C. R. Fisher, M. C. Kennicutt II, J. M. Brooks, Science 247, In addition to the implications for more bility of larvae finding a suitable home, 1094 (1990). 10. J. J. Robinson, J. L. Stein, C. M. Cavanaugh, J. Bacteriol. energy-efficient carbon fixation, this finding it is likely that a high percentage of the 180, 1596 (1998). may help explain the anomalously high nutritional input from the symbionts goes 11. K. M. Scott, Environ. Microbiol. 5, 424 (2003). carbon isotope values that have puzzled directly to reproduction. How is this ac- researchers for decades. complished and coordinated? Furthermore, 10.1126/science.1137739

MOLECULAR BIOLOGY Small RNA molecules that silence gene expression are amplified by different Amplified Silencing mechanisms in nematodes and plants. David C. Baulcombe

en years ago, we knew nothing about ence to control gene expression in biotech- the target sites of the primary silencing RNA. how double-stranded RNA blocks gene nology and for understanding the effects of RNA-directed RNA polymerases (RdRPs) Texpression through the silencing of silencing RNAs on cell function and organ- produce secondary siRNA, and the new re- targeted RNA. We now have a good under- ism development. sults indicate that they catalyze two different standing of this process, and current interest is Specifically, these new studies investigate mechanisms of silencing amplification. One turning to variations on the basic mechanism. how the target of silencing can spread (or tran- mechanism is characterized by Axtell et al. Recent studies involving plants and the nema- sit) within a single strand of RNA. The initia- (3), who investigated endogenous secondary tode Caenorhabditis elegans continue this tor of transitivity is a double-stranded RNA siRNAs in plants. They show that efficient sec- trend, including those reported in this issue by that is first processed by Dicer, a ribonuclease ondary siRNA production occurs if a single- Pak and Fire on page 241 (1) and Sijen et al. III–like enzyme, into short interfering RNA stranded RNA has two target sites for the on page 244 (2). Two other papers by Axtell et (siRNA) or a related type of RNA referred Argonaute ribonucleoprotein. Optimal sec- al. (3) and Ruby et al. (4) are also relevant. to as microRNA (miRNA). These 21- to 25- ondary siRNA production occurs when the These studies deal with the amplification nucleotide single-stranded RNAs are the pri- targeted RNA is cleaved by Argonaute. Cleaved of silencing-related RNA and explain how mary silencing RNAs in the transitive process. RNA then recruits RdRP, which generates strong, persistent silencing can be initiated A primary silencing RNA binds to a ribonu- double-stranded RNA. Dicer then produces with small amounts of “initiator” double- clease H–like protein of the Argonaute transitive secondary siRNAs (see the figure). stranded RNA. The amplification process has class. The resulting Argonaute ribonucleopro- Another biogenesis mechanism of second- implications for application of RNA interfer- tein can target long RNA molecules by ary siRNAs has, so far, only been described in Watson-Crick base pairing. The targeted C. elegans. The discovery of this distinct RNA then becomes a source of secondary mechanism by Sijen et al., Pak and Fire, and The author is in the Sainsbury Laboratory, John Innes Centre, Norwich NR4 7UH, UK. E-mail: david.baulcombe@ siRNAs. Transitivity occurs when the second- Ruby et al. follows from the observation sainsbury-laboratory.ac.uk ary siRNAs correspond to regions adjacent to that a type of siRNA is underrepresented in

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