Feature Interdomain Interactions: Dissecting Animal–Bacterial Symbioses Downloaded from https://academic.oup.com/bioscience/article/56/5/376/234704 by guest on 01 October 2021

MELISSA LEE PHILLIPS

or the first billion years of life on FEarth, prokaryotes had the place to themselves. By the time the first eukary- otes and then, finally, multicellular or- ganisms arose and diversified, prokaryotic bacteria and archaebacteria had invaded almost every nook of the planet and in- vented nearly every known metabolic pathway. “All the animal body plans evolved in a microbe-rich marine environment,” Immature individual of the spittlebug Clastoptera arizonana, showing says developmental biologist Margaret the brightly colored bacteriomes on each side of the abdomen. Two McFall-Ngai of the University of different symbionts live in this insect: Candidatus Sulcia muelleri and an Wisconsin–Madison.“It’s very likely that unnamed species of ß-proteobacteria. Photograph: Phat Tran. it was a very common thing to associate with microorganisms.” toxin that wards off wolf spiders, and to offspring through the egg cell, while Selective pressure from other organ- several insects have symbionts that con- bacteria that live outside host cells are isms has come not only from creatures fer resistance to extreme heat. usually picked up from the environment evolving alongside them but, in many Some symbiotic bacteria colonize host by each new generation of animals. In cases, from inside of them. Virtually all epithelial tissue, such as the mammalian some animal species, adults participate animals are thought to have some type of intestine and termite hindgut, while oth- directly in the microbial colonization of symbiotic prokaryote living inside them, ers have actually invaded host cells and juveniles: Adult termite workers feed their although these relationships can span a live almost as organelles. A few animals, feces to newly hatched juveniles, pre- vast continuum from fleeting to requisite including deep-sea shrimp and nema- sumably to ensure the juveniles’ hindguts and from beneficial to pathogenic. todes that live in coastal sediments, pos- are exposed to the correct consortia of Prokaryotes can influence host biology sess permanent bacterial coats on the bacteria. and evolution in incredibly diverse ways. outside of their bodies. A host’s reliance on its symbionts also Luminescent bacteria form light organs The site of colonization in the host spans a wide spectrum, from pathogens in many species of fish, possibly for dif- tends to correlate—although not that the host’s body actively tries to elim- ferent evolutionary reasons. Bacteria absolutely—with how the symbiont is inate to obligate symbionts, without called Wolbachia are reproductive para- transmitted from generation to genera- which the host dies. Insects that feed ex- sites in insects but obligate mutualists in tion. Intracellular symbionts are fre- clusively on restricted diets, such as plant nematodes. Beetles’ symbionts secrete a quently passed vertically from mother sap, blood, or wood, often receive essen-

376 BioScience • May 2006 / Vol. 56 No. 5 www.biosciencemag.org Feature tial nutrients from their symbiotic part- ners and cannot reproduce without them. Many animals also possess an array of fac- ultative symbionts, whose composition may change frequently. Relationships are obligate or facultative from the symbionts’ point of view, as well: Vertically transmitted bacteria that provision their hosts with essential nu- trients often have such degraded genomes that they could not hope to survive out- side a host. Many horizontally transmit- Downloaded from https://academic.oup.com/bioscience/article/56/5/376/234704 by guest on 01 October 2021 ted symbionts, however, have a free-living stage in which they fend for themselves. The Hawaiian bobtail squid, Euprymna scolopes, contains a light organ Biologists have often been reluctant filled with luminescent bacteria called Vibrio fischeri. The squid–Vibrio to consider how such associations have af- relationship is one of the best-studied animal–bacterial symbioses. fected the evolution and development of Photograph: Margaret McFall-Ngai, University of Wisconsin–Madison. the animals they study, says McFall-Ngai. “On occasion, they will think about how environmental pressure will influence other, and scientists have developed ex- host gene expression that alter the mor- development, and they think about tem- perimental genetic systems in both or- phology of the light organ. Bacterial mor- perature and pressure and osmolarity,” ganisms that permit control over genes of phology and gene expression also change says McFall-Ngai. “Rarely do they think interest. upon establishment of the symbiosis. about biotic pressure.” A similar swap of genetic messages The symbionts alter their surface com- This neglect stems at least somewhat appears to occur in one of the most ponents and the signal molecules they from technological difficulties in study- successful models of animal–bacterial export, both of which encourage the sym- ing microorganisms. Laboratory cultures symbiosis: the association between the biosis to move forward. are difficult for many free-living bacteria Hawaiian bobtail squid, Euprymna Molecular genetic manipulation of V. and most likely impossible for those scolopes, and its luminescent bacterial fischeri has allowed scientists to find bac- living completely dependent existences symbiont, Vibrio fischeri.The V. fischeri terial genes required to establish, pro- inside animals. But new and improving live inside the squid’s light organ and mote, and maintain the symbiosis, technologies, especially in high-throughput emit light of the same color and intensity McFall-Ngai says. She and others are now DNA sequencing and genomics, are al- as moonlight and starlight, thereby pre- working on techniques involving anti- lowing biologists to survey symbiotic in- venting the nocturnal squid from casting sense probes and viral vectors to knock teractions as never before. a dark shadow visible to predators or down expression of certain genes in the prey below. squid to see how that affects symbiosis de- Experimenting with symbioses Like the legume–rhizobium sym- velopment. Much of what is known about symbioses biosis, the squid–vibrio symbiosis is Another animal system that promises between prokaryotes and eukaryotes binary—one symbiont, one host. And, to reveal more about the development of comes from the study of leguminous although Euprymna normally become animal–bacterial symbioses is the germ- plants and a group of nitrogen-fixing colonized with free-living V. fischeri free mouse model. Observations of mice bacteria called rhizobia. These bacteria fix within minutes to hours after they hatch raised in sterile environments can reveal atmospheric nitrogen for the host plant as juveniles, they can be kept sterile past how the microbial community as a whole in exchange for carbon and protected this stage in the lab and later infected contributes to mammalian development housing in the plant’s root nodules. These at various ages. Such experiments have and physiology. Adding one or a few nodules develop over weeks of back-and- revealed much about how these part- members of the normal gut microbiota forth gene expression between plant and ners interact and influence each other’s back into a germ-free mouse’s gastro- bacterium, creating a conversation that biology. intestinal tract can also elucidate those begins with each partner recognizing the During embryogenesis, developing microorganisms’ specific roles in the other and results in the morphogenesis of squid acquire a ciliated tissue whose sole community. the root nodule. purpose appears to be to promote later Beginning at birth, mammals amass Legume–rhizobium symbioses have bacterial colonization. Squid mothers incredibly complex microbial consortia made ideal experimental models for sev- lay their eggs in areas enriched for V. in many body regions, especially in the in- eral reasons, says McFall-Ngai: They in- fischeri, and after symbionts colonize the testine. A human gut, for example, carries volve only one host and one symbiont, hatchlings, both partners undergo major up to a thousand species of mainly anaer- both partners can be cultured without the changes. The bacteria induce changes in obic microorganisms, which together en- www.biosciencemag.org May 2006 / Vol. 56 No. 5 • BioScience 377 Feature compass about 10 times as many cells as sess obligate intracellular symbionts that are in the entire human body. provide the host with essential nutrients. Mammals’ relationship with intesti- These “primary” symbionts live inside nal microbiota was probably driven by a specialized host cells called bacteriocytes, nutritional need, just as in the vast ma- which make up a dedicated organ in the jority of animal–bacterial symbioses, says anterior gut, called the bacteriome. Many Lora Hooper, a molecular biologist at of these insects also have “secondary” the University of Texas Southwestern symbionts that live outside the bacteri- Medical Center in Dallas. ome. A well-established example of this Ribosomal RNA analyses in sap- symbiosis is found in ruminants like feeding aphids and their primary sym- Downloaded from https://academic.oup.com/bioscience/article/56/5/376/234704 by guest on 01 October 2021 cows, sheep, and deer. These animals biont Buchnera aphidicola have shown are able to digest grass because bacteria that Buchnera infected an ancestor of all of today’s aphids between 200 and in their rumen break down cellulose The digestive tract of the medicinal 250 million years ago, says biologist into simple carbohydrates. This nutri- leech, Hirudo medicinalis,is Nancy Moran of the University of Ari- tional need was likely the driving force dominated by symbionts called zona in Tucson. Since then, aphids and behind the evolution of mammal– Aeromonas veronii biovar sobria. Buchnera have evolved in parallel: Each microbiota partnerships, Hooper says, The leech–Aeromonas lineage of Buchnera has passed verti- but millions of years of coexistence have symbiosis is a much simpler system to cally from mother to offspring, and the resulted in microbiotic control over study than digestive-tract symbioses two organisms have diverged and spe- many host functions in addition to in mammals. Photograph: ciated together. carbohydrate processing. Joerg Graf, University The theme of ancient infection fol- For example, germ-free mouse exper- of Connecticut. lowed by parallel paths of speciation and iments have clearly shown that a normal An early success of microbial genomic divergence has been reported for other microbiota is indispensable for the de- analysis was the verification of mito- animal species that harbor vertically velopment of healthy immune and di- chondria and chloroplast origins. transmitted, obligate symbionts, Moran gestive systems in mammals. Recent Brought to the attention of the scientific says, including tsetse flies, cockroaches, studies have also shown that the micro- community in the 1970s by Lynn Mar- carpenter ants, deep sea clams, and ne- biota affects fat metabolism, enhances gulis of the University of Massachusetts– matodes. epithelial healing, and can even trigger de- Amherst, the theory that these eukaryotic To find out more about a species than velopment of blood vessels in the gut. organelles began as free-living bacteria its place on a genetic tree, however, sci- “It was really the first study that sug- was extremely controversial—until ge- entists have developed techniques to iden- gested that normal gut microbes could netic analysis confirmed the close relat- tify genes other than rRNA—without trigger a morphogenetic event—actually edness between DNA sequences found in the need to culture bacteria or amplify changing gut anatomy,”Hooper says. organelles and those in free-living bac- known genomic regions. Such studies Hooper’s group is trying to under- teria that exist today. Since then, advances were termed “metagenomics”by Jo Han- stand how the immune system corrals in cloning and sequencing technologies delsman of the University of Wisconsin– members of the microbiota within the in- have allowed previously unimagined ge- Madison and her colleagues in 1998. Al- testine and keeps them from crossing netic reconstructions of symbiotic micro- though the term usually implies studies into other parts of the body, where they organisms that cannot survive outside of microbial communities containing could cause inflammation or sepsis, she their hosts. multiple species, the same techniques says. Microbial species that cannot be grown can be applied to sequencing isolated in culture are usually first identified by symbionts that cannot live outside their Genomic—and metagenomic— sequencing of their ribosomal RNA hosts, Handelsman says. DNA fragments approaches (rRNA) genes. Every cellular organism are simply cloned from raw samples and Experimental models of symbiosis have contains these genes, and they evolve assembled into libraries. From there, sci- their limits. In no animal system can sci- slowly and predictably, which makes entists can conduct functional analyses of entists manipulate both host and sym- them good evolutionary clocks. rRNA the fragments or mass-sequence them— biont genetics as they can in legumes gene typing can tell scientists how closely and, in some cases, reconstruct a com- and rhizobia, McFall-Ngai says. To add to microbial species are related to one an- plete genome. knowledge gained from these models, other and where they belong on a bacte- In 2000, Buchnera became the first un- evolutionary biologists are using genomic rial phylogenetic tree. cultivated intracellular symbiont to have analyses of symbiotic prokaryotes to Phylogenetic analyses have proved ex- its genome completely sequenced. Earlier understand how they interact with their tremely fruitful in the analysis of insect studies of the aphid–Buchnera symbiosis hosts. symbionts. Many types of insects pos- had revealed that Buchnera most likely

378 BioScience • May 2006 / Vol. 56 No. 5 www.biosciencemag.org Feature provides aphids with essential amino acids absent from their phloem sap diets, says evolutionary biologist Jennifer Wernegreen of the Marine Biological Laboratory in Woods Hole, Massachu- setts. But sequencing gave unprecedented insight into the genome of an obligate in- tracellular symbiont and into the inter- twined biochemistries and evolutionary histories of a host and its symbiont. Millions of years of evolution inside an Downloaded from https://academic.oup.com/bioscience/article/56/5/376/234704 by guest on 01 October 2021 animal host have changed the genomic landscape of Buchnera in dramatic ways. At around 650 base pairs, it is the small- est bacterial genome known—seven times smaller than that of its closest free- living relative, Escherichia coli. Buchnera has lost many genes essential to free- living bacteria, including those involved in DNA repair, homologous recombina- tion, biosynthesis of cell-surface com- ponents, and defense of the cell. A major part of the genome is devoted to synthe- sizing essential amino acids for the host, but genes for synthesizing nutrients that aphids can make have been lost. Soon after Buchnera invaded an aphid ancestor, its genome appears to have gone Electron micrograph of Buchnera cells within an aphid bacteriocyte. Buchnera have through major changes, including large provided aphids with essential nutrients for more than 200 million years. deletions and chromosomal rearrange- Photograph: J. White. ments. But in the millions of years that followed, its DNA sequence has been re- missed, even in the cultured organisms,” maximum of a dozen species of bacteria markably stable. Loss of genes involved in she says. in its gut, so “we’re hoping that would be recombination and gene transfer proba- If strain-level heterogeneity is wide- a target for reconstruction of genomes,” bly underlie this stability in part, Werne- spread among symbiotic organisms, how- Handelsman says. green says. This pattern seems to be ever, it could make fitting together Even when genetic sequences are characteristic of many symbionts’ evo- genomes a difficult task, says Handels- known, however, gene function can only lution, as seen in the completed genomic man, especially if one sample contains be deduced for sequences that bear re- sequences of the symbionts of tsetse flies, DNA not only from different strains of semblance to genes in previously culti- carpenter ants, and a nematode, among the same species but from many different vated sister species. To get at the functions others. species. That’s the ultimate goal of of completely novel gene sequences, A likely future target for genome metagenomics—to reconstruct genomes which Handelsman estimates usually reconstruction is the archaebacterium from a mix of DNA fragments from dif- make up about 60 percent of metage- Cenarchaeum symbiosum, which lives ferent prokaryotic organisms. nomics libraries, scientists can screen dif- inside the marine sponge Axinella mex- This type of complex analysis—which ferent DNA fragments to see how they icana. Scientists have cloned genomic is also performed on consortia of bacte- function in E. coli or another cultivable fragments from the symbiont, and se- ria found in soil and elsewhere in the host.“We figure that, if you’re ever going quence analysis has revealed that two environment—hasn’t resulted in the re- to understand what’s really going on, we very closely related species of Cenar- construction of an entire genome yet, have to move beyond what we can rec- chaeum live together in the same although Handelsman is optimistic that ognize by sequence similarity,”Handels- sponges—an unexpected finding that will happen soon. One problem, she says, man says. would never have been discovered with is that there are very few intermediate traditional sequencing techniques, Han- cases between binary symbioses and an Symbiotic diversity delsman says. “It’s starting to make us extremely complex gut. Her lab, how- A pilot genome reconstruction project realize how much strain variation we’ve ever, studies a gypsy moth that carries a currently under way holds promise for www.biosciencemag.org May 2006 / Vol. 56 No. 5 • BioScience 379 Feature peering into a chemically different kind it possesses no digestive system. In bi- of symbiosis: the alliance between mouth- valves, which store their symbionts in less, gutless marine invertebrates and gill epithelial cells, the division of labor is bacteria that produce energy without not quite as clear. Giant clams have ves- light from the sun. tigial feeding appendages, Cavanaugh In 1980, microbial ecologist Colleen says, which suggests their ability to feed Cavanaugh and her colleagues reported is reduced but may not be absent, while that the rich, diverse ecosystems at hy- mussels have been shown to take up par- drothermal vents contain a previously ticles from their environment. unknown type of symbiosis between After discovery of hydrothermal vent deep-sea tubeworms and their sulfur- symbiotic communities, chemosynthetic oxidizing bacterial symbionts. symbioses were found in other places Downloaded from https://academic.oup.com/bioscience/article/56/5/376/234704 by guest on 01 October 2021 Many chemosynthetic bacteria—so where oxic and anoxic environments called because they acquire energy to meet, including coastal sediments and build organic compounds from chemical hydrocarbon seeps. In coastal sediments reactions—oxidize hydrogen sulfide gas live nematodes with symbiotic chemo- and use the resulting energy to fix carbon synthetic bacteria on the outside of their dioxide into organic carbon compounds bodies and mouthless, gutless oligo- that feed their animal hosts. Others chaete worms with up to six different acquire both energy and carbon by oxi- species of symbiotic bacteria living ex- dizing methane. Some animals, such Giant tubeworms (Riftia pachyptila) tracellularly under the worm’s cuticle. as Atlantic mussels, have been found to living at hydrothermal vents have no Coastal nematodes and oligochaetes contain both sulfur- and methane- mouth or gut and rely completely on spend time in the sulfide-rich sediments oxidizing symbionts. symbiotic bacteria for nutrition. These but also come to the surface for oxy- Chemosynthetic bacteria thrive at bacteria can turn hydrogen sulfide, gen, fulfilling both requirements of their hydrothermal vents because chemosyn- methane, and carbon dioxide into symbionts. “They act like little eleva- thesis requires reduced chemicals like organic molecules that feed the worm. tors for the bacteria,”Cavanaugh says. sulfides and methane, which are found in The worm reciprocates by supplying The giant clam and its symbionts have hydrothermal vent fluid, as well as oxy- chemical nutrients for the bacteria. been shown to cospeciate, just as in gen, which is abundant in the surround- Photograph: Eric DeChaine, aphid–Buchnera symbioses. Symbiont ing seawater. Oxygen and reduced Harvard University. genetic material has also been detected chemicals “are typically available in mu- in the ovaries of the coastal clam Sole- tually exclusive habitats,”says Cavanaugh, mya velum, which suggests that these of Harvard University, which means the bacteria are vertically transmitted. En- bacteria must either access both envi- vironmental transmission of symbionts ronments themselves or live inside a host is generally inferred from a lack of sym- that will access both for them. biont DNA in the ovaries as well as from While animals like mussels can move nonidentical phylogenies between host through these different environments, and symbiont. Vestimentiferan tube- the sessile tubeworm body actually spans worms of multiple genera all share a the divide between oxic and anoxic zones. similar symbiont, which suggests that In addition, the tubeworm possesses an this symbiont is passed horizontally extracellular hemoglobin that binds oxy- among different species. gen the same way it does in most animals, Genome sequencing projects will un- while it also binds sulfur in a different cover more about chemosynthetic sym- molecular site. This specialized hemo- biont genomics, as well as about how far globin nourishes the tubeworm’s sym- The orange-red color of the Caribbean each partnership is “on the evolutionary bionts and also colors the bright red great star coral Montastraea path from being two separate, free-living plume that emanates from the worm. cavernosa comes from fluorescent organisms,” Cavanaugh says. Sequenc- In return for access to its energy sub- cyanobacteria that live in its cells. ing is planned for four chemosynthetic strates, the bacterium provides organic These cyanobacteria fix nitrogen, symbionts: a tubeworm and giant clam, compounds to sustain its host. Symbionts which is a limiting element for the both from the deep sea, and two species of the tubeworm Riftia pachyptila,which coral. The coral also hosts symbiotic of coastal clams. In each environment, live inside bacteriocytes in an organ called dinoflagellates (zooxanthellae). one animal is thought to acquire sym- the trophosome, are thought to provide Photograph: Hays Cummins, bionts vertically and the other horizon- 100 percent of the animal’s nutrition, as Miami University. tally, Cavanaugh says, which should

380 BioScience • May 2006 / Vol. 56 No. 5 www.biosciencemag.org Feature maximize the diversity of symbioses the coral and its symbiotic zooxanthellae also genes appear to control infection by my- sequences will represent. benefits from the nitrogen-fixing activi- corrhizal fungi and by nitrogen-fixing A future target for many types of sym- ties of cyanobacteria inside the host coral rhizobia, even though these symbioses biosis studies may be figuring out how cells. And in January 2006, a group from involve very different functions and were multiple prokaryotic symbionts in one the University of Kansas reported that established at very different times in evo- host have evolved and function together. what was thought to be an ancient tri- lutionary history, Handelsman says. “I Little is known about the molecular path- partite relationship among ants, fungi, think that one of the themes that will ways that allow bacteria and animal cells and fungal parasites actually involves a emerge, and maybe is emerging, is that, to communicate, Moran says, and equally fourth member: antibiotic-producing at different points in evolution, differ- little is known about how different bacteria that live in cavities on the in- ent genes or different molecules get kind prokaryotic symbionts—such as the ar- sects’ bodies and help ward off the par- of co-opted for different purposes,” she ray of bacteria and archaea found in asites. The entire group has probably says. “That’s becoming one of the more Downloaded from https://academic.oup.com/bioscience/article/56/5/376/234704 by guest on 01 October 2021 many intestines—may talk to each other. been evolving together for at least 10 interesting directions of the field.” Also, recent studies have revealed other million years, the scientists estimate. types of multiple-partner symbioses in Organisms involved in these multiple- unexpected places. In 2004, a group of kingdom symbioses may even use the Melissa Lee Phillips (e-mail: [email protected]) New England biologists reported that a same genes to control relationships with is a freelance science writer based known partnership between a Caribbean different partners. In plants, common in Seattle, Washington.

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