FEATURE ARTICLE

Cant Live without You: Essential Animal-Bacterial Relationships

The resident bacteria that animals require contribute metabolic capabilities and signaling networks that shape host health
Angela E. Douglas

For billions of years, the bacteria (Eubacteria and Archea) had this planet to themselves. This exclusivity changed with the origin of the eukaryotes, little more than 1 billion years ago. In one of the greatest turning points in the history of life, eukaryotic cellular organization permitted greater biological complexity than has evolved in bacteriaat every level from the individual cell to multicellular organisms with divisions of labor and social interactions among organisms. Although the diversity of both eukaryotes and bacteria is predominantly at the unicellular level, only the eukaryotes include large radiations of organisms that interact with the environment at spatial scales of 1 cm or more. The success of the eukaryotes would not have been self-evident at their evolutionary inception. The proto-eukaryote lacked key features that contributed to the biological success of bacteria, especially the many metabolic innovations that enable bacteria to use a wide range of energy sources and to thrive under a great variety of environmental conditions. The success of the eukaryotes has depended utterly on their ancientand continuingcapacity to associate with bacteria. All modern eukaryotes bear (or have evolved from eukaryotes that bear) an erstwhile -proteobacterium, the mitochondrion. Without this association, the eukaryotic radiation would have been in the energetic slow lane, excluded from the aerobic habitats that had been created through the innovation of oxygenic photosynthesis in cyanobacteria and colonized by a diversity of bacteria with aerobic respiration. Right from the start, the eukaryotes have had a cant live without you relationship with bacteria. What did the ancestor of the mitochondria gain from this relationship? Arguably, the large size of the proto-eukaryotic cell and its capacity

to accommodate membrane-bound inclusions was crucial to the relationship. The eukaryote was a novel habitat with the resources to support the bacterium that provided for aerobic metabolism. As a consequence, the mitochondrion may be the most successful bacterial lineage. The cooperative relationship between eukaryotes and the bacteria that evolved into mitochondria is not unique. Again and again, eukaryotes enhanced their impoverished metabolic capabilities by entering into symbiotic associations with bacteria. Symbioses of great ecological and evolutionary importance include the eukaryotic acquisition of photosynthesis by the ancestors of eukaryotic algae and plants and of nitrogen fxation, for example between plant roots and rhizobia. A particularly rich diversity of associations with bacteria is found among the animals. The animals and their immediate ancestors compounded their second-rate metabolic inheritance as eukaryotes by the evolutionary loss of addi-

SUMMARY
The success of eukaryotes, which arose more than 1 billion years ago, depends very much on their continuing capacity to associate with bacteria. The microbial ancestor of mitochondria, which provides aerobic metabolism for the host eukaryotic cell, may be the most successful bacterial lineage. The resident bacteria contribute to the metabolic, regulatory, and morphogenetic networks of animals, optimizing function and contributing to host health. The impact of obligate vertical transmission on bacterial symbionts in various animalsfor instance, among sap-feeding aphidsis profound and typically includes loss of redundant genes encoding metabolic functions. Learning how to correct imbalances when host animals and their microbiota are misaligned may lead to medical strategies for alleviating various diseases.
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FIGURE 1

The bacterial symbiosis in aphids. (A) The pea aphid Acyrthosiphon pisum. (B) Fluorescence in situ hybridization (FITC: green) of Buchnera cells in bacteriocytes, located in the body cavity. (C) Transmission electron micrograph of the coccoid Buchnera cells in the cytoplasm of bacteriocytes. [Micrographs by Calum Russell (A); Simon Chandler (B); and Angela Douglas (C)]

tional capabilities. On multiple occasions, these missing capabilities proved valuable to exploit certain habitats or diets, and animals reacquired them, not by metabolic evolution but by associating with bacteria or eukaryotic microorganisms that possess the needed trait. Animals as a group lack the capacity to synthesize 9 or 10 amino acids that make up protein (the essential amino acids) and many of the coenzymes required for function of enzymes central to metabolism (such as B vitamins). Some animal groups have lost additional metabolic capabilities, including sterol synthesis in insects and other arthropods and cellulose degradation in vertebrates. As a result, animals that feed through their lifecycle on blood (defcient in B vitamins) or plant sap (defcient in essential amino acids) cant live without microorganisms, usually bacteria, that possess these biosynthetic capabilities. Most vertebrate herbivores require cellulolytic microorganisms which degrade the plant cell wall polysaccharides to products utilizable by the animal. In addition, associations with autotrophs have enabled animals to exploit nutrient-poor, shallow-water habitats (corals bearing photosynthetic algae) and zones of mixing between oxic and anoxic habitats at hydrothermal vents, methane seeps, and in marine sediments (various invertebrate animals bearing chemosynthetic bacteria).
Mutual Dependence, Sharing of Metabolic Functions Are Key in Symbioses

In many animal-bacterial symbioses, both animal and bacteria cannot live without their partner.
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This mutual dependence is particularly evident in relationships with obligate vertical transmission, meaning that the bacteria are transferred from mother to offspring animal, usually via the egg, and have no free-living phase. Associations with obligate vertical transmission are very prevalent in insects, especially groups living on the nutritionally unbalanced or poor diets of vertebrate blood and plant sap. Sap-feeding aphids and plant phloem are one such association (Fig. 1). The impact of these associations on the evolutionary history of the bacterial symbionts has been profound, involving two interacting processes. One process is the evolutionary transition from selfsh metabolism to cooperative metabolism (Fig. 2A), such that the bacteria synthesize certain nutrients at suffcient rates to meet both their own requirements and those of the animal host. For example, 50% or more of the essential amino acids synthesized by the -proteobacterium Buchnera aphidicola in aphids is released to the host, supporting aphid growth and reproduction. This evolutionary transition was accompanied by coevolved changes in the host, to supply the bacteria with substrates that enable the selective overproduction of essential amino acids. Arguably, this metabolic specialization has been facilitated by the evolution of a novel cell type that functions to house and maintain the symbionts. The cell, generically called a bacteriocyte (see Fig. 1B), evolved independently in various different insect groups. Transcriptomic and proteomic analyses of the aphid bacteriocyte show that genes with enriched expression in the bacteriocyte have a predominantly metabolic function,

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FIGURE 2

Coevolutionary processes shaping insect-bacterial symbioses with obligate vertical transmission. (A) Transition from selsh to cooperative traits in symbiotic bacteria. (B) Genome degradation in symbiotic bacteria.

especially in providing the precursors that Buchnera require for essential amino acid synthesis. The second coevolutionary change involving vertically transmitted bacterial symbionts and their animal hosts relates to genome reduction of the bacteria and associated functional compensation by the animal (Fig. 2B). Obligate vertical transmission leads to genome reduction partly because the loss of a free-living life stage results in relaxed selection and decay of genes that are redundant in the host habitat. Compounding this process is the small effective population size of the bacteria, arising from population bottlenecking at vertical transmission. If all the bacteria transferred to an individual host bear a mutation that is mildly deleterious, the mutation can persist and, furthermore, select for compensatory responses in the host. As a consequence of relaxed selection and genomic deterioration, many bacteria inhabiting insect bacteriocytes have a genome size smaller than 1 Mb, and possess very restricted functional capabilities that largely complement the metabolic capabilities of their host. In some instances, the animal and bacteria have different linked metabolic pathways, and in others they have different reactions within a single pathway. For ex-

ample, purine metabolism in the aphid-Buchnera symbiosisis partitioned between the partners: the aphid is capable of purine synthesis on its own but lacks key enzymes in purine salvage that are present in other insects, while Buchnera has retained the ancestral -proteobacterial pathways for the salvage, but not synthesis, of purines (Fig. 3A). Buchnera also lost the genetic capacity for certain reactions in the synthesis of essential amino acids. Notably, the terminal reaction in the synthesis of branched-chain amino acids (BCAsisoleucine, leucine, and valine) by bacteria is mediated by the enzyme branched-chain amino acid aminotransferase (BCAT). Animals also possess BCAT, where it generally functions in the reverse direction as the frst step in BCA degradation. In aphids, BCAT, but no other enzyme in the pathway for BCA degradation, is enriched within bacteriocytes. This enrichment, together with the evolutionary loss of the capacity to mediate BCAT reactions in Buchnera, leads to the prediction that the pathway for BCA synthesis is shared between the two partners: the penultimate metabolite in BCA synthesis is transferred from Buchnera to the bacteriocyte, where the BCAs are synthesized and then partitioned between the two partners (Fig. 3B).
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FIGURE 3

Complementary metabolic capabilities of the animal host and bacterial symbiont in the pea aphid-Buchnera symbiosis. (A) Complementary pathways in purine metabolism. The animal host (black arrows) mediates synthesis of nucleotides (GMP, guanosine monophosphate; XMP, xanthosine monophosphate; IMP, inosine monophosphate; AMP, adenosine monophosphate) de novo from PRPP (phosphoribosyl pyrophosphate) and synthesis of nucleosides (guanosine, xanthosine, inosine and adenosine). Buchnera utilizes aphid-derived guanosine and hypoxanthine to generate its nucleotide requirement by purine salvage pathways (red arrows). Buchnera cannot salvage adenine, which is released to the aphid and recycled by the aphid to AMP. (B) Complementary reactions in branched chain amino acid synthesis. Buchnera mediates all the reactions apart from the terminal reaction, which is mediated by aphid BCAT (branched-chain amino acid transaminase).

Why Animals Require Bacterial Partners

The exquisite coevolutionary interactions between some animals and bacteria, as illustrated by the relationship between aphids and Buchnera, offer satisfying explanations for why certain animals cannot live without their bacterial partners. The failure of aphids experimentally deprived of Buchnera to grow or reproduce can be attributed to the multiple ways in which the metabolism of the partners is coupled. Buchnera differs from mitochondria in that functional Buchnera genes have not been transferred to the host nucleus, and Buchnera function is apparently not subsidized by proteins of nucleo-cytoplasmic origin. Nevertheless, these types of interactions are particular to certain animals, and they cannot explain why all animals cant live without bacteria. Most animals do not bear obligately vertically transmitted bacteria (apart from the bacteriallyderived mitochondria), but they are copiously colonized by bacteria that can account for 10% or more of their biomass.
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Most bacteria associated with animals are not deleterious. In fact, axenic animals, which are reared under bacteria-free conditions, are unhealthy. Growth and developmental rates are reduced in axenic Drosophila fruit flies; laboratory mice are profoundly unhealthy, with impaired development of their gastrointestinal tract and immune system; and axenic zebrafsh die prematurely from progressive degeneration of the epidermis. Why? The dependence of these animals on bacteria cannot be attributed entirely to specifc functions of the bacteria, equivalent to essential amino acid synthesis by Buchnera. Instead, it appears that the metabolic, regulatory, and morphogenetic networks of animals function optimally in the context of inputs from the resident bacteria. For example, the slow development of axenic Drosophila has been linked to low insulin signaling: specifc metabolites released by bacteria in the gut act as signals that modulate insulin signaling, apparently to the optimal level for host growth and development. Currently, major research programs are devoted to understanding how the resident microbiota shape animal

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and human health, with exciting opportunities for novel medical applications to alleviate the burden of various chronic diseases.
Imbalances between Bacteria and Their Animal Hosts: Health Implications

The basis of the animal-bacterial interactions that defne the healthy animal lies in the deep evolutionary history of animals. All animals evolve and live in the context of a resident microbiota. Microbial products may have provided cues for environmental conditions that favored particular patterns of gene expression, cellular behavior, or developmental events in early animals and their ancestors, and thereby contributed elements of the signaling or other networks that regulate animal functions. While some of the bacterial effects may relate to environmental factors relevant to modern animals, others may have been retained as signatures of interactions past because their elimination would have been disruptive to the complex regulatory networks of the animal. Furthermore, the selective interests of the animal and its microbiota are not perfectly aligned, and the microbiota may manipulate the host to enhance nutrient supply or alter the growth, reproductive, or behavioral patterns to their own advantage. These manipulations may, in turn, have selected for compensatory responses in the animal host. Such responses are likely constitutive: because the microbiota are never absent under natural conditions, there is no selection for function without the manipulative microbial effects. There will be impaired animal function in the absence of the microbiota, even where the

microbiota provide no function beyond its impact on the set point of host metabolism, immune system, or other processes. Such dependence without evident beneft can be described as addiction. Cant live without you accurately describes the half-billion-year relationship between animals and bacteria. Animal dependence on bacteria may have multiple bases: a requirement for specifc capabilities of bacteria, the signature of interactions past in which an animal function requires input from the bacteria, and addiction to the manipulative traits of bacteria. All three types of interaction are advantageous to the bacteria in that they provide coevolved bacteria with tickets to colonize their animal hosts.
Angela E. Douglas is the Daljit S. and Elaine Sarkaria Professor of Insect Physiology and Toxicology in the Department of Entomology, Cornell University, Ithaca, N.Y.

Suggested Reading
Aanen, D. K., and R. F. Hoekstra. 2007. The evolution of obligate mutualism: if you cant beat em, join em. Trends Ecol. Evol. 22:506 509. Douglas, A. E. 2011. Lessons from studying insect symbioses. Cell Host Microbe 10:359 367. Shin, S. C., S. H. Kim, H. You, B. Kim, A. C. Kim, K. A. Lee, J. H. Yoon, J. H. Ryu, and W. J. Lee. 2011. Drosophila microbiome modulates host developmental and metabolic homeostasis via insulin signaling. Science 334:670 674. Smith, K., K. D. Mccoy, and A. J. Macpherson. 2007. Use of axenic animals in studying the adaptation of mammals to their commensal intestinal microbiota. Semin. Immunol. 19:59 69.

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