Genomic Insights Into Prokaryote–Animal Symbioses

REVIEWS Nature Reviews Genetics | AOP, published online 12 February 2008; doi:10.1038/nrg2319

Learning how to live together: genomic insights into prokaryote–animal symbioses

Andrés Moya*‡, Juli Peretó*§, Rosario Gil*‡ and Amparo Latorre*‡ Abstract | Our understanding of prokaryote–eukaryote symbioses as a source of evolutionary innovation has been rapidly increased by the advent of genomics, which has made possible the biological study of uncultivable endosymbionts. Genomics is allowing the dissection of the evolutionary process that starts with host invasion then progresses from facultative to obligate symbiosis and ends with replacement by, or coexistence with, new symbionts. Moreover, genomics has provided important clues on the mechanisms driving the genome-reduction process, the functions that are retained by the endosymbionts, the role of the host, and the factors that might determine whether the association will become parasitic or mutualistic.

Symbiosis Prokaryotic microorganisms are widespread in all the symbioses during the origin and evolution of eukaryotic 7 From the Greek, sym ‘with’ and environments on Earth. Given their ecological ubiquity, cells, although controversies about the details persist . biosis ‘living’. A long-term it is not surprising to find many prokaryotic species in In addition, on the basis of its wide distribution across association between two or close relationships with members of many eukaryotic major phylogenetic taxa (BOX 1), symbiosis could have more organisms of different species that is integrated at the taxa, often establishing a persistent association, which an important role in the evolution of species that are behavioural, metabolic or is known as symbiosis (BOX 1). According to the fitness involved in such partnerships. In all the major biological genetic level. According to effects on the members of the symbiotic relationship, phenomena that are classified as symbiotic, new cellular the level of dependence on the associations can be referred to as parasitism, mutualism structures and/or metabolic capabilities emerge as a host, symbiosis can be obligate or commensalism and, depending on the location of the result of evolutionary forces, favouring the maintenance or facultative. The term was 8 introduced by Anton de Bary symbiont with respect to host cells, as ectosymbiosis or of the association . The interplay of both partners or, in and Albert Bernard Frank when endosymbiosis. Among intracellular symbioses, there are some cases, between a single host and more than one discussing lichens and differences regarding the extent of dependence between symbiont, forms an evolving biological community that mycorrhizae, respectively, at the animal host and the symbiont and the age of the asso- changes throughout time. the end of the 1870s. ciation, leading to the dichotomic classification between In most cases of symbioses between prokaryotes and obligate primary endosymbionts (P-endosymbionts) eukaryotes, the relationship between host and symbiont *Institut Cavanilles de and facultative secondary symbionts (S-symbionts). is so close that the microorganisms cannot be cultured, Biodiversitat i Biologia P‑endosymbionts generally have long evolutionary histo- making them difficult to study. However, in the past Evolutiva, Universitat de ries with their hosts, whereas S‑symbionts seem to have few years, genome sequencing, transcriptomics and València, Apartado de correos metagenomics 22085. 46071 València and established more recent associations, retaining the ability the recently emerged field of — which 1–4 CIBER de Epidemiología y to return to a free-living condition . In some cases, an avoid having to culture microbial cells — have offered Salud Pública, Spain. S‑symbiont can evolve to become an obligate partner new opportunities for symbiosis research. Whole ‡Departament de Genètica and and, if a P‑endosymbiont is already present, microbial genomes of several intracellular bacterial symbionts §Departament de Bioquímica i Biologia Molecular, consortia can be established. have been sequenced, allowing comparisons among Universitat de València, For several decades, the idea that microbial associa- the evolutionary innovations of these bacteria, from Dr. Moliner, 50. 46100 tions might be central to eukaryote evolution remained being free living to various stages of integration with Burjassot (València), Spain. controversial. However, the work of Lynn Margulis since their respective hosts9–23 (TABLE 1). Such genomics Correspondence to A.M. the late 1960s not only contributed to the establishment approaches to the study of symbiosis will allow several e-mail: [email protected] 5 doi:10.1038/nrg2319 of a symbiotic theory for cell evolution , but also revived questions to be tackled, contributing to an understand- 6 Published online earlier proposals that were forgotten by biologists . ing of the evolutionary relevance of this phenomenon. 12 February 2008 Today, there is a wide consensus on the essential role of In particular, these questions relate to the nature of the

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Box 1 | Spread of symbiosis in the biosphere Stable symbioses have independently evolved many times in diverse recycling. In the best-studied cases — those of symbioses involving groups of eukaryotes. Although the scope of this review is restricted to insects97 — the presence of such associations throughout most of insect prokaryotic partners that are associated with animal hosts, there are many evolutionary history suggests that symbiosis has been a driving force in other remarkable examples of symbiosis between eukaryotic organisms, the diversification of this group. as exemplified by fungal associations with other fungi, protists, plants The figure shows the phylogenetic distribution of symbioses, indicating (that is, mycorrhizae), algae (that is, lichens) and animals (for an overview, the bacterial and archaeal classes within which there are associations see ref. 90). Most symbioses have a proven biochemical foundation. In with eukaryotic hosts. Data were collected from the literature and are some cases, one of the partners benefits from compounds that are the result of a long tradition of studies that have used ecological, produced by the other; in other cases, the waste products of one partner developmental, morphological, biochemical and genomic approaches to (especially nitrogen compounds) are recycled by the other. investigate symbiosis (a list of examples is given in the Supplementary The eukaryotic nucleocytoplasm has limited metabolic capabilities. The information S2 (table)). The uneven distribution of symbiosis highlights mitochondrial respiratory chain and oxygenic photosynthesis in the diverse interests and motivations of the scientific community. the chloroplast are two examples of metabolic functions that have been Advances in genomics have had a large impact on the research into acquired through symbiotic associations with prokaryotic partners microbial symbioses, which has implications for biotechnology (for during early eukaryotic evolution8. Countless symbiotic relationships example, sponge-associated prokaryotes98), agriculture (for example, have emerged more recently, or are in the process of developing, and nitrogen fixation in plant-associated bacteria91) and biomedicine (for they complement the limited metabolic networks of most eukaryotes example, the human intestine microbiome99). Metagenomic methods with several prokaryotic metabolic capabilities, such as dinitrogen have notably increased our knowledge of the biodiversity of non- fixation91, methanogenesis92, chemolithoautotrophy93, nitrogen cultivable bacteria in symbiotic consortia, including the description of assimilation94 and essential-nutrient anabolism95,96. In particular, animal completely new candidate phyla — Endomicrobia100 and Poribacteria101. heterotrophic metabolism is relatively narrow and some amino acids, Postulated symbiotic events leading to the evolutionary origin of vitamins and fatty acids must be obtained from an external source. Thus, organelles (mitochondria and chloroplasts) are indicated. The branching symbiotic associations with microorganisms have allowed animals to order in the eukaryotic phylogenetic tree has been adapted from refs adapt to specialized feeding behaviours by providing the nutrients that 102,103. The phylogenetic tree of animals is an adaptation from ref. 104, are deficient in their restricted diets. In fact, all intracellular mutualistic modified by recent molecular phylogenies105,106. The lengths of branches symbioses between bacteria and animals that have been analysed at the are not to scale. An asterisk indicates that complete genomes are genomic level9–23 (TABLE 1) are related to nutrient provision and waste available (see TABLE 1).

Cy Cy Rhizaria Cy, α, β, γ, δ, M a nt ozoa ra a rc fe at ko Ce Excavata Fi, S, M ol ro ramini a ve Fo Metamonada Al Hete Cy, , M β Euglenozoa, Radiolari A, Cy, α, β, γ Heterolobosea Viridiplantae Malawimonas Amoebozoa Rhodophyta Plantae E Oxymonada Discosea Glaucophyta A, B, Ch, Fi, α, β, γ, ε Variosea Jakobidae Ch, γ Lobosea Chloroplast Mycetozoa Cryptophyta M Archamoebae Haptophyta Opisthokonta Cristidiscoidea B, F, Fi, γ, M Centrohelid heliozoa Vertebrata Cy Fungi Cy, γ Urochordata Choanozoa Apusozoa Cephalochordata Animalia Mitochondria Echinodermata Eukaryotes Hemichordata Xenoturbellida Prokaryotes Priapulida α*, γ Nematoda Archaea Arthropoda Methanobacteria M A, B*, Fu, S, α, β, γ*, δ, ε, M Thermoprotei T B, α, γ, δ, ε, S Annelida Bacteria γ Ectoprocta Sipunculida Acidobacteria Ac Nitrospira N γ*, ε Mollusca Actinobacteria A α-Proteobacteria α Rotifera Bacteroidetes B β-Proteobacteria β Platyhelminthes Chlamydiae Ch γ-Proteobacteria γ Ctenophora Chloroflexi Cl δ-Proteobacteria δ Cy, α, β Cnidaria Cyanobacteria Cy ε-Proteobacteria ε Fibrobacteres F Spirochaetes S Porifera Firmicutes Fi Verrucomicrobia V Ac, A, Cl, Cy, Fi, G, N, α, β, γ, δ, S, V, P, T Fusobacteria Fu Ca. Endomicrobia E Gemmatimonadetes G Ca. Poribacteria P Nature Reviews | Genetics nature reviews | genetics advance online publication | 219 © 2008 Nature Publishing Group

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Table 1 | Genomic data for mutualistic symbionts of animals Organism Host Metabolic Genome GC content CDS rRNAs tRNAs Pseudogenes Accession mode size (kb) (%) number Buchnera Acyrthosiphon pisum Heterotroph 652 26.24 574 3 32 12 BA000003, aphidicola BAp* (aphid)‡ AP001070, AP001070 Buchnera Schizaphis graminum Heterotroph 653 26.3 556 3 32 33 AE013218, aphidicola BSg* (aphid) AF041836, Z21938 Buchnera Baizongia pistaciae (aphid) Heterotroph 618 25.3 507 3 32 9 AE016826, aphidicola BBp* AF492591 Buchnera Cinara cedri (aphid) Heterotroph 422 20.2 362 3 31 3 CP000263, aphidicola BCc* AY438025 Blochmannia Camponotus floridanus Heterotroph 706 27.4 583 3 37 4 BX248583 floridanus*§ (carpenter ant) Blochmannia Camponotus Heterotroph 792 29.6 610 3 39 4 CP000016 pennsylvanicus*§ pennsylvanicus (carpenter ant) Wigglesworthia Glossina brevipalpis Heterotroph 698 22.5 617 6 34 14 BA000021, glossinidia* (tsetse fly) AB063523 Sodalis Glossina morsitans Heterotroph 4,171 54.7 2,516 7 69 972 AP008232, glossinidius* (tsetse fly)‡ (Secondary) AP008233, AP008234, AP008235 Baumannia Homalodisca coagulata Heterotroph 686 33.2 595 6 39 9 CP000238 cicadellinicola*§ (sharpshooter) Sulcia muelleri§|| Homalodisca coagulata Heterotroph 245 22.4 227 3 31 – CP000770 (sharpshooter) Carsonella ruddii*§ Pachypsylla venusta (psyllid) Heterotroph 160 16.6 182 3 28 – AP009180 Wolbachia wBm¶ Brugia malayi (nematode)# Heterotroph 1,080 34 805 3 34 98 AE017321 Ruthia magnifica*§ Calyptogena magnifica Autotroph 1,200 34.0 976 3 36 – CP000488 (Deep-sea clam) Vesicomyosocius Calyptogena okutanii Autotroph 1,000 31.6 937 3 35 – AP009247 okutanii*§ (Deep-sea clam) Nitratiruptor sp** Deep-sea-vent animals Autotroph 1,878 39.7 ~1,118 9 45 ~739‡‡ AP009178 Sulfurovum sp** Deep-sea-vent animals Autotroph 2,563 43.8 ~1,218 9 44 ~1,248‡‡ AP009179 The data shown are for those symbionts that have been sequenced as of January 2008. Data were retrieved from the National Center for Biotechnology Information (NCBI). *γ-Proteobacteria. ‡Genome sequence in progress. §These bacteria are called Candidatus. ||Bacteroidetes. ¶α-Proteobacteria. #Complete genome available, see ref. 78. **ε-Proteobacteria; although it is unclear whether these two isolates are epibiotic symbionts or another variation of symbionts, many genome features strongly support their symbiotic lifestyle. ‡‡Estimated from ref. 20. CDS, coding sequences; rRNA, ribosomal RNA.

genome-reduction process, the type of genes that are aspects such as metabolic interdependencies between retained by the endosymbiont, the molecular pathways partners and the survival of the prokaryote in the face that are used by the host to control the endosymbiont of the host immune response. population and the complex set of factors that determine whether the final outcome of the association is Genomics coverage of prokaryotic endosymbionts parasitic or mutualistic. Obligate endosymbionts of animals. In recent years, Here we focus on the study of mutualistic endosym- the genomes of several bacterial P‑endosymbionts of Parasitism (TABLE 1) Symbioses in which one bioses of animals for which genomic studies have been animals have been completely sequenced . species is increasing its fitness carried out, providing new biological and evolutionary P‑endosymbionts are inherited by strict vertical trans- while the fitness of the other insights. Although only one host genome has been mission and, in most cases, reside in specialized host species is adversely affected. sequenced, complete genomic sequences for several cells called bacteriocytes. Functional analyses have cor-

Mutualism bacterial symbionts are available. Comparisons with roborated the nutritional role of the associations; each Symbioses in which both free-living relatives have revealed dramatic changes in bacterium provides the nutrients that are deficient in species increase their fitness. genome size and content, and have begun to reveal the the diet of its animal-host, whereas the bacterium gains mechanisms by which these changes take place and their a permanent supply of a wide range of metabolites that Commensalism functional consequences. Furthermore, the mechanisms are provided by the host. Symbioses in which one partner is increasing its fitness that are involved in the establishment, maintenance and Most of the endosymbionts that have been studied without affecting the other evolution of mutualistic associations are being scruti- are γ‑proteobacteria that live in obligate association species. nized; this has led to the emergence of new insights into with insects, and they provide various functions to

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their host, including: the provision of essential amino cedar-aphid endosymbionts (FIG. 2), led Pérez-Brocal acids9,10,12–14,16,21,23, vitamins and cofactors9–12,15 that are and co-workers21 to conclude that S. symbiotica SCc absent in the host diet; nitrogen recycling and storage13,14; might have the potential to replace B. aphidicola BCc. and the provision of metabolic factors that are required In a second example, genomic sequencing has been for survival and fertility19. Whereas most of these species carried out for Baumannia cicadellinicola and the are heterotrophic endosymbionts, two recently sequenced Bacteroidetes species Sulcia muelleri, the co-resident Ectosymbiosis endosymbionts that are harboured by deep-sea clams of P‑endosymbionts of the xylem-feeding sharpshooter A symbiosis in which the the genus Calyptogena17,18 are chemolithoautotrophic, and Homalodisca coagulata15,23. Genomic analysis has symbiont lives on the body surface of the host, including provide almost all nutrients to the host by fixing CO2 revealed that the two symbionts have complementary internal surfaces such as the and using H2S as a source of energy and reducing power biosynthetic capabilities, which are needed to provide lining of the digestive tube and (FIG. 1 and Supplementary information S1 (figure)). their host with nutrients that are lacking in xylem sap. the ducts of glands. Phylogenetic analysis revealed that Sulcia was ancestrally Facultative symbionts. Occasionally, hosts tolerate present in a host lineage that acquired Baumannia at the Endosymbiosis Symbioses in which a S‑symbionts that coexist with a P‑endosymbiont and same approximate time as the switch to xylem feeding, 24 prokaryote symbiont lives that can be either deleterious or beneficial to the host . consistent with the view that its nutrient-provisioning inside a eukaryotic cell. In aphids, a number of facultative symbionts that reside capabilities were a requirement for the host to evolve in multiple host tissues, cells surrounding primary towards this lifestyle41. As in the case of Buchnera– Primary endosymbiont (P-endosymbiont). Obligate bacteriocytes or in their own bacteriocytes have been Serratia, the two symbionts live in close proximity within 25 15 bacterial endosymbionts that described . Although they are normally vertically trans- the host bacteriome . live inside specialized animal mitted, their distribution patterns suggest that sporadic Metagenomic approaches have also been used to host cells called bacteriocytes. horizontal transmission among host individuals and host analyse more complex consortia, including the four co- The association is obligate for species must have occurred26. Among the possible occurring symbionts from the segmented worm Olavius both partners. beneficial roles that have been identified, some of these algarvensis42. In this instance, the host belongs to a group 27–29 Secondary symbiont S‑symbionts can rescue the host from heat damage , of oligochaetes that lack a mouth, gut and anus, and (S-symbiont). Facultative provide defence against natural enemies (parasitoids and that are unique among annelids in having reduced their bacterial endosymbiont pathogens)30–32 and participate in host specialization33–36. nephridial excretory system. O. algarvensis harbours that coexists with a γ ( P‑endosymbiont. Often In other cases, facultative symbionts can spread among at least four symbionts, two -proteobacteria sulphur located in syncitial cells near lineages without conferring a benefit, by manipulating oxidizing) and two δ-proteobacteria (sulphate reducing), ref. 37 the bacteriocyte and in various host reproduction (reviewed in ). For example, which fix CO2 and provide the host with nutrients. Almost other insect tissue types. in Wolbachia infections of arthropods, the symbiont all amino acids and several vitamins can be synthesized by Secondary symbionts are not undergoes transfer among host lineages. Remarkably, the symbionts and then provided to their host, probably essential for host survival and are transferred horizontally Wolbachia can also be a P‑endosymbiont in filarial through controlled digestion of the bacteria as suggested 19 43 among individuals of both the nematodes , indicating that the same prokaryotic line- by the vacuole localization of the host . The symbionts host species and other species. age can take part in more than one type of symbiotic are also likely to be involved in host-waste recycling. association. Metagenomics Genomic changes during endosymbiont evolution The application of genomic At present, the only completely sequenced genome analyses to uncultured of an S‑symbiont is that of Sodalis glossinidius (TABLE 1). Genomic changes experienced by endosymbionts. A gen- microorganisms. Also referred This bacterium coexists in the gut lumen of tsetse flies eral feature of intracellular pathogenic and mutualistic to as environmental genomics. with the P‑endosymbiont Wigglesworthia glossinidia, bacteria is that they have smaller genomes with a higher although they occupy different areas of the gut and can AT content than their free-living relatives44. The causes Vertical transmission 22 The endosymbionts are be found both intra- and extracellularly . It has been of reductive-genome evolution are related to both the maternally transferred, that suggested that S. glossinidius has an important role in genetic information that is needed in the new environ- is, directly from a host to the acquisition of trypanosome infections38. ment and to population dynamics, although the relative its offspring. importance of these factors has not been determined45,46.

Bacteriocytes Endosymbiotic consortia. Although the importance of The change from a free-living environment to one that is Specialized cells of the host syntrophy between unrelated organisms has been recog- intracellular and protected implies that many genes are species in which symbiotic nized in several cases39, the new field of metagenomics rendered unnecessary, whereas others become redun- bacteria live. provides the ability to study bacterial endosymbionts dant because their functions can be supplied by the host. that coexist in the same host, enabling the discovery Therefore, some genetic material can be lost without a Heterotrophy The metabolic mode in which of complex and stable associations, and analysis of the detrimental effect, and can accumulate mutations owing the carbon source is organic contribution of each partner to the relationship. to the lack of effective natural selection to purge them. In matter. By extension, this is a One endosymbiotic consortium that has recently addition, owing to the strict vertical transmission of obli- metabolic mode in which been reported involves Buchnera aphidicola BCc gate endosymbionts, only a few bacteria are transmitted, organic matter is the source of carbon, electrons and energy and Serratia symbiotica SCc, which coexist in the which generates continuous bottlenecks that favour the 40 (chemoorganoheterotrophy). bacteriome of the aphid Cinara cedri . S. symbiotica is action of random genetic drift. The combination of these a facultative symbiont in other aphid species, but has factors allows the accumulation of mutations in genes Chemolithoautotrophy been found in all the cedar-aphid populations that that can be beneficial but that are not essential — such The metabolic mode in have been analysed, casting doubts over its faculta- as genes involved in DNA repair and recombination which CO2 is the carbon source and an inorganic tive status in this species. Functional and evolutionary (see below) — further increasing the repertoire of genes chemical reaction is both the comparative analyses of the sequenced B. aphidicola that can be lost, and reducing the possibility of genetic electron and energy source. genomes, as well as microscopic analysis of the two exchange by homologous recombination. Furthermore,

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a CO2

+ + + + Nucleus + Ion transport PTS Nutrient of host transport cell to the host Glycolysis Mitochondria Candidatus Nitrogen Ruthia magnifica metabolism Reductive Cell-wall pentose pathway biosynthesis Membrane- phospholipid Protein Inner and outer biosynthesis Amino-acid biosynthesis export ATP (except Thr and Ile) stem bacterial membranes sy

Sulphur Sec metabolism Fatty-acid De novo biosynthesis nucleotide biosynthesis H2S and salvage Cofactor biosynthesis pathways Electron (except pantothenate and UQ) transport + H ATP H+ Membrane- O 2 associated Cytoplasm of Vacuole of the ATP synthesis the host cell host cell

b Reduced carbon, non-essential amino acids

+ + + + PTS Nucleus + Ion transport of host ? Nutrient cell transport Glycolysis to the host Mitochondria Buchnera Nitrogen aphidicola BCc metabolism Non-oxidative pentose pathway Protein Cell-wall export

biosynthesis Membrane- stem phospholipid Amino-acid biosynthesis sy biosynthesis Inner and outer (except Trp) Sec bacterial membranes

Sulphur De novo metabolism Fatty-acid nucleotide biosynthesis? biosynthesis Flagella and salvage export Cofactor biosynthesis pathways apparatus Electron (except riboflavin) transport H+ Membrane- O2 Horizontal transmission Cytoplasm of associated ATP synthesis Some endosymbionts retain a the host cell Serratia symbiotica generalized ability to colonize Vacuole of the host cell and persist in multiple hosts, that is, their transmission is Figure 1 | Comparative biochemistry of symbiont–host interdependence. As a study case, we use information from between individuals of the Nature Reviews | Genetics same or different host species, complete genome sequences to infer and compare the metabolisms that are encoded by the reduced genomes of an rather than from parent to autotrophic and a heterotrophic endosymbiont. Blue indicates the presence of a metabolic process, orange indicates its 17 offspring. absence. a | Candidatus Ruthia magnifica uses electrons from H2S to drive an autotrophic metabolism based on the reductive pentose phosphate pathway (Calvin–Benson cycle), supplies essential nutrients (including amino acids and Syntrophy cofactors) to its host, and recycles nitrogen. As is the case in the Calyptogena okutanii endosymbiont18, it has the potential Emergence of new metabolic for anabolism of all essential biomolecules except threonine (Thr), isoleucine (Ile) and ubiquinone (UQ). The missing steps capabilities as a result of in those pathways could be explained by the function of an unidentified enzyme, or by complementation with the host symbiosis, it is often metabolism. b | Buchnera aphidicola BCc uses reduced-carbon molecules and non-essential amino acids to fuel its essential for the survival heterotrophic metabolism and synthesize essential nutrients, with the exception of tryptophan (Trp) and riboflavin21. of the consortium. These molecules could be provided by a second co-existing endosymbiont, Serratia symbiotica. The self-sufficiency of Bacteriome R. magnifica sharply contrasts with the incomplete metabolic abilities of B. aphidicola BCc. Among the few similarities An organ-like structure formed between both metabolisms, note the absence of nutrient-transport systems to the host. A more detailed version can be by bacteriocytes. found in the Supplementary information S1 (figure). PTS, phosphotransferase system; Sec system, secretory system.

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the intracellular environment isolates the symbiont from elements. The increase in frequency of these elements in other bacterial species and thus reduces, and can even the newly established P‑endosymbiont must be due to eliminate, the possibility of gaining new genetic mate- an increase in the replicative transposition of elements rial by horizontal gene transfer. Thus, gene losses can that were resident at the onset of symbiosis24. All analysed become irreversible. endosymbiotic bacteria that are involved in older associa- In addition to having reduced genomes, an increased tions possess genomes that are free of mobile elements. AT content is another general feature of the endosymbi- Presumably, therefore, the expansion of mobile ont genomes that have been sequenced so far. The bias elements must at some point have become deleterious towards AT is probably due to the loss of genes for DNA and they must have been removed as part of the process of repair, leading to a mutational GC to AT pressure47 (for genome degradation. The difference in the abundance a metabolic hypothesis see ref. 48). Such a bias has a of transposable elements in two strains of Wolbachia notable effect on many genes by altering the structure with different lifestyles and genome sizes supports this and function of the corresponding proteins. In fact, it statement. In Wolbachia pipientis wMel57, a reproductive has been proposed that GroEL, a chaperonin that is parasite of Drosophila melanogaster, 14% of the 1.27-Mb constitutively overexpressed in B. aphidicola, helps to symbiont genome is occupied by repetitive DNA and post-translationally correct the altered structure of many mobile elements, whereas in W. pipientis wBm19, the obli- proteins that is caused by this bias49. A by-product of the gate endosymbiont of the nematode Brugia malayi, these base-composition bias of these genomes is the loss of elements account for just 5.4% of the 1.08-Mb genome. the codon-usage bias that is typical of free-living bacte- During the second stage of the genome-reduction ria: it is almost absent from B. aphidicola, and is highly process, genome shrinkage seems to have mostly reduced in P‑endosymbionts with larger genomes and occurred through a process of gradual gene loss, scat- in S‑symbionts50,51. tered along the genome. Such loss seems to follow The degree of both genome-size reduction and a pattern that starts with the inactivation of a gene increase in AT content vary among endosymbionts, (pseudogenization) by single-nucleotide mutations, and correlate with the age of the association. P‑endosymbionts that are partners in old associations a have generally smaller genomes and an AT content higher than 70%, whereas S‑symbionts and P‑endosymbionts 10 µm S that are part of younger associations have genome sizes and AT contents intermediate with respect to older n 2,25,50 P‑endosymbionts and free-living relatives . P P n The genome-reduction process examined. To understand the different stages of the genome-reduction process it is necessary to analyse a range of genome sequences, from S endosymbionts that still have genomes similar in size to those of their free-living relatives, to those that have undergone the most dramatic reductions. Such comparative analyses suggest that the process of genome shrink- P age might have taken place in two separate stages. S A massive gene loss must have occurred soon after the establishment of the obligate symbiosis, probably by b means of large deletions that would cause the elimination of a series of contiguous genes52. The accumulation of mobile elements, representing a source for chromosomal rearrangements and gene inactivation, seems to have an m important role at this first stage. This is suggested by m the fact that mobile elements are relatively abundant in bacteria that have recently acquired an obligate, intracellular way of life53,54. Mobile elements, such as bacterial insertion sequences (IS), are fairly abundant in the S‑symbiont S. glossinidius22, but even more so (estimated Figure 2 | Bacteriocytes of the aphidNatur Cinarae Reviews cedri | Genetics. at more than 25% of genome content) in its close rela- a | A 1.5 mm semi-thin section that shows both primary tives SOPE (Sitophilus oryzae primary endosymbiont) and secondary bacteriocytes, easily identifiable by their different tones under toluidine blue staining. and SZPE (Sitophilus zeamais primary endosymbiont). b | Electron micrographs of the same individual that These species are P‑endosymbionts of the rice and maize show the round shape of Buchnera aphidicola and weevils, respectively, and both have recently established Serratia symbiotica, respectively, in their bacteriocytes. obligate associations with their hosts55,56 (sequencing of P, primary symbiont (B. aphidicola); S, secondary symbiont the SOPE genome is in progress). (S. symbiotica); n, nuclei of both bacteriocytes; These data indicate that a common symbiotic ancestor m, mitochondria. Electron micrographs courtesy of of these two lineages must already have possessed these A. Lamelas, Universitat de València, Spain.

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and continues with a rapid reduction in length until the required for host survival, as we discuss later. Another original gene is completely eroded58,59. Even in advanced general feature of all sequenced endosymbiont genomes stages of the reductive process, genome-length reduc- is the loss of most DNA repair and transcriptional regulation is mainly due to the loss of protein-coding genes, tory mechanisms. Furthermore, losses affect most genes not to their shortening21. Regarding intergenic regions, of the latter category, indicating that there is no need there is a slight size reduction in the four sequenced for transcriptional regulation in a stable environment. B. aphidicola strains when compared with their close This hypothesis is supported by the results of microarray relative Escherichia coli60. The shortening of intergenic experiments in B. aphidicola62,63, which show constitu- regions is more evident in extremely reduced genomes, tive gene expression of metabolic genes, regardless of as observed in Carsonella ruddii16, in which this has changes in environmental conditions. been so extreme as to have led to overlapping genes. Other features of the functional content of endo- In C. ruddii, the annotated ORFs are also consider- symbiont genomes are more dependent on their spe- ably shorter than their orthologues in other bacteria, cific lifestyles. For example, the bacterial cell envelope although the functionality of these shortened ORFs is seems to be highly simplified, being less structured in questionable61. bacteria living inside host-derived vesicles (for example, B. aphidicola) than in endosymbionts that live free in Functional changes in hosts and symbionts the cytosol of bacteriocytes (for example, Blochmannia The first step towards the establishment of an obligate floridanus and W. glossinidia)13. The simplification can endosymbiosis takes place when a free-living bacterium be extreme; for example, B. aphidicola BCc has lost all infects the host. Then, both organisms co-evolve to adapt the genes that are involved in the biosynthesis of the to the new situation. On the bacterial side, genomics bacterial cell wall21. studies have revealed that the endosymbiont genome gets smaller during this adaptive process, owing to the Metabolic changes and nutrient transport. The bigger loss of genes that are rendered unnecessary in the new endosymbiont genomes, belonging to more recently environment; however, a symbiotic bacterium must still established partnerships, have retained many genes that retain a number of functions to allow its continued sur- are involved in several intermediary metabolic path- vival in the host. By contrast, our understanding of how ways, but most of these have been lost in the smaller the animal host is affected at the genetic level, and how it genomes of well-established endosymbionts (FIG. 1; has solved the problem of having a bacterium (or more TABLE 1). The loss of metabolic genes is also affected to a than one) inside its cells, is poorly understood. However, certain extent by the presence of other bacterial species host specialization implies that it is now dependent on in the intracellular environment of the symbiont, in that the bacteria to survive and reproduce. Adaptive changes the simultaneous presence of S‑symbionts can compen- in the host include the development of specialized cells sate for the metabolic deficiencies of the P‑endosymbiont, where bacteria reside, adequate systems to control bacte- as well as those of the host64. rial populations, plus the modification of its immune Even though each endosymbiont (or the combina- response against the intruder cells. tion of symbionts within a consortium) has specifically retained the pathways that are necessary to synthesize General changes in endosymbiont gene content. The the nutrients that respective hosts are unable to provide catalogue of individual genes that are shared by all for themselves, endosymbionts exhibit limited transport analysed endosymbionts suggests that the molecular capabilities65 (FIG. 1). This situation has been observed in mechanisms that are necessary for survival in an intra- B. aphidicola, the two deep-sea-clam endosymbionts cellular environment share broad commonalities across that were recently analysed17,18 and the endosymbionts of all endosymbiotic associations13. Only about one-third O. algarvensis 42. The subcellular localization of endo- of the coding capacity of each endosymbiont genome symbionts inside vacuole-like compartments66,67, and that has been sequenced so far is devoted to the require- the presence of lysozyme in the host cells68,69, strongly ments of the specific symbiosis, with these genes mainly suggest that, in these systems, the host nutritional needs reflecting differences in host lifestyle, nutritional needs could be satisfied by controlled weakening or killing of and location within host cells. symbiont cells — a case of a necrotrophic host–symbiont Across the sequenced endosymbiont genomes, genes relationship. that are involved in essential functions — such as those involved in DNA replication, transcription and transla- Invasion strategies: have pathogens been domesticated? tion — are more likely to be retained than are genes Recent studies of the mechanisms that are used by endo- of other functional classes, and sometimes account for symbionts to establish and maintain their infection of more than one-third of the genome21. Chaperone sys- host tissues indicate that invasion strategies are based on tems and all essential components of the protein trans- the same molecular tools that are used by well-studied location machinery are also retained in these genomes, pathogenic bacteria (reviewed in refs 24,47). These ensuring the correct folding and localization of protein mechanisms include the use of various secretion systems components9,49. for the attachment and invasion of host cells64 and the Although gene losses affect all functional categories, utilization of the same quorum-sensing mechanisms they are nonrandom. The most dramatic losses affect for the regulation of virulence or mutualistic traits, genes that are involved in metabolism but are not depending on the type of association17,18.

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Cap violaceum, respectively. Interestingly, both regions have a FliD complete set of genes encoding a functional T3SS export apparatus, but they lack many genes encoding effector proteins in the orthologous pathogenic islands. Moreover, Hook–filament junction Filament the only putative effector proteins that are preserved are a FlgK FlgL FliC specifically involved in the cytoskeletal rearrangements Hook necessary for bacterial cell invasion24. FliK FlgD FlgE Both parasitic and mutualistic Wolbachia lack a T3SS, but they do possess a complete set of genes for another bac- FlgH ring L Rod FliE FlgB FlgC FlgF FlgG terial secretion system, T4SS, which is used by many path- Outer membrane ogens to secrete proteins in order to invade host cells19,57. FlgI ring P B. aphidicola and W. glossinidia — well-established Cell wall Basal P‑endosymbionts in an advanced stage of genome reduc- body Stator MotA MotB Inner membrane tion and with highly simplified cell envelopes — also lack Export apparatus a canonical T3SS, but still retain surface mechanisms for FliF MS ring FlhA FlhB FliOP FliQ FliR exporting proteins, such as a simplified flagellar appara- tus71 (FIG. 3). Because these structures are quite abundant ATPase Rotor (ring C) Flil FliH FliM FliN FliG at the B. aphidicola surface — even though these are non- FliJ motile bacteria72 — and only those elements homologous FlgN to the T3SS have been retained in all of these reduced FlgA genomes21 it has been proposed that these structures b FlgH FlgF must be involved in invading new bacteriocytes, ovaries and embryos to ensure transmission to host offspring9. FlgI The immune response and control of bacterial invasion. The molecular mechanisms that are involved in the host immune response to bacterial invasion remain FlhA FlhB FliOP FliQ FliR FliF poorly understood73 but, clearly, hosts have adapted to maintain rather than eliminate endosymbionts. But how FliI FliH FliN FliG does the host immune system perceive these bacteria and control their growth and invasion without complete Figure 3 | The flagellum, an example of genome reduction at theNatur structurale Reviews level. | Genetics a | The Escherichia coli flagellum. Protein and structural components are indicated: cap, bacterial clearance? filament (formed by ~20,000 copies of flagellin), hook–filament junction, hook and basal Hosts have developed molecular systems to recognize body. The basal body is formed by a set of rings for anchorage to the inner and outer conserved microbial cell-envelope motifs (for example, membranes and to the cell wall, the rod, the stator, the rotor and the export apparatus peptidoglycan) through receptors such as those that were (including one ATPase). b | Vestiges of the flagellar apparatus in Buchnera aphidicola identified following the whole-genome sequencing of BCc. Elements that are involved in the formation of the flagellar filament and hook, the D. melanogaster74. Endosymbionts that have an ancient stator/motor and the transcriptional regulators are lost. None of them is necessary for a relationship with their hosts have highly simplified cell non-motile bacterium. Many elements that are involved in the anchorage of the export envelopes and, therefore, these old associations are not apparatus to the cell envelope have also been lost, consistent with the absence of a well- good models to study this type of immune control. structured outer membrane and cell wall. However, the MS‑ring protein (FliF), two out of However, analyses of younger endosymbiotic associa- three components of the C‑ring (FliN and FliG), the P‑ring protein (FlgI) and L‑ring protein (FlgH) are present. Only one protein for the rod formation (FlgF) is preserved. tions provide insights into the early stages of the interplay All the retained proteins are homologous to the components of the type III secretion between host immunity and bacterial virulence, for exam- system, supporting the hypothesis that this structure is used by the bacterium as a ple, endosymbionts of weevils from the genus Sitophilus. protein export system to help in the invasion of new host cells71. Heddi et al. carried out an in vivo broad characterization of the transcriptional response of the bacteriocyte to intracellular bacteria, using SZPE as a model organism75. Sitophilus spp. P‑endosymbionts, and their close They found that the bacteriome expresses a peptidog- relative the S‑symbiont S. glossindius, encode presum- lycan recognition protein (PGRP) that is homologous ably functional genes of the type III secretion system to the PGRP-LB protein of D. melanogaster, a catalytic (T3SS)70, an indication that the presence of the secretion member of the PGRP family. The amidase activity of system pre-dates the origin of the symbiotic association. PGRP-LB reduces the biological activity of peptidoglycan, The T3SS is a crucial virulence factor of many gram- and therefore downregulates the host immune response negative pathogens of plants and animals, including against gram-negative bacteria76. The specific role of the humans. However, it also seems to be a beneficial factor, weevil PRPG is still under investigation, but the fact that Pathogenic island enabling the establishment of a mutualistic, intracel- it contains the amino-acid residues that are responsible A part of a genome, for lular insect–bacteria association. In the S. glossinidius for amidase activity, and that PRPG transcripts only which there is evidence of genome there are three regions that contain distinct accumulate when endosymbiotic bacteria are present its acquisition by horizontal 77 transfer, that encodes genes T3SS systems. The best characterized are SSR‑1 and in the bacteriome , indicate that it might work in the that contribute to the virulence SSR‑2, which contain genes that are related to genes same way, thus — reducing the host’s defence against of a pathogen. found in Salmonella enterica and Chromobacterium the endosymbiont to allow the long-term interaction.

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Only one sequenced genome is available for a eukary- the intracellular bacteria to their host. This scenario otic host of an endosymbiont that also has a sequenced was initially proposed in the 1930s by Lwoff 81 and has genome — the genome of B. malayi 78, the nematode host been confirmed by genomic studies (see previous sec- of Wolbachia wBm. Consistent with a mitigated immune tion). However, the bacterial genome, no matter how response to control rather than eliminate the endosym- small, must retain the genes that are essential for the biont, the immune system of B. malayi has lost the ability maintenance of the symbiotic relationship (for example, to encode the small antibacterial peptides described in essential nutrient provision), and a reduced repertoire of other sequenced nematodes79. The genome sequence genes for self-maintenance, self-reproduction and evo- of the aphid Acyrthosiphon pisum will soon be avail- lution. Otherwise, genome reduction might eventually able and will provide us with additional clues into how lead these bacteria towards extinction. hosts control bacteria in the later stages of the symbiotic In time, a second symbiont can join the consortium. association. Although initially this new association is likely to be One additional insight into the control of endosymbi- facultative, a new stable relationship can be established, onts by their hosts comes from the finding that endosym- in which case the two bacterial species will co-evolve. bionts that do not reside in host-derived vesicles, but that As the genome-reductive process continues, and genes live free inside the cytosol, have lost the gene dnaA11,13. that are shared with the second endosymbiont become This gene encodes a protein that is involved in the ini- redundant, two possible outcomes can occur. The tiation of DNA replication, which might indicate that actual outcome is a matter of chance, depending on there is a direct control of bacterial growth by the host. which genome is affected by the loss of genes that are In the case of B. aphidicola, which does reside in host- needed for the synthesis of molecules that are essential derived vesicles, the high level of lysozyme expression for fitness. detected in bacteriocytes68,69 might be an important fac- One possibility is that the presence of a second symbi- tor involved in the host control of bacterial abundance, ont might accelerate the degenerative process of the first, as this enzyme catalyses the hydrolysis of peptidoglycan leading to its extinction and replacement by the formerly — a system normally used by animals as a constitutive facultative symbiont, which will then become obligatory. defence against bacterial pathogens. This replacement has been reported in symbioses of some weevils56 and might also be taking place in the case of The evolution of specialized host cells. Using transcrip- the Buchnera–Serratia consortium that has been estab- tomic analysis, a pioneering study was carried out into lished in the aphid C. cedri 21. Unlike other sequenced the development and evolution of aphid cells that are B. aphidicola strains, BCc has partially lost its symbiotic destined to become bacteriocytes. Several transcription role, as it cannot synthesize tryptophan and riboflavin factors were found to be expressed in these cells early (see Supplementary information S1(figure)), which need on in development — before colonization by the symbi- to come from another source, not only for the survival ont population acquired from the mother — following of the host, but also for that of B. aphidicola. Genes from a pattern that has not been described in any other insect S. symbiotica SCc that are involved in the biosynthesis cells80. Even when bacteria are experimentally removed of tryptophan have been identified, revealing that this by antibiotic treatment, bacteriocytes are still specified species might be the source of this essential amino acid. and maintained. The main conclusion of this work is The complete sequence of S. symbiotica SCc (currently that bacteriocytes represent an evolutionarily novel cell in progress) will tell us whether this bacterium is able to fate, which is developmentally determined independ- perform all the metabolic functions needed to maintain ently of the presence of bacteria. Regarding the role its host’s fitness, or whether it has also lost some path- of the bacteriocyte itself in the symbiotic relationship, ways. In the first case, extinction of the P‑endosymbiont the identification of a number of bacteriocyte-specific, (Buchnera) and replacement by Serratia would be the highly expressed genes in the aphid A. pisum by tran- most plausible hypothesis. scriptome analysis69 revealed that most of them were Alternatively — highlighting the second possibil- involved in several functions: non-essential amino-acid ity for the evolutionary outcome of a second symbiont metabolism, consistent with this process being among establishing itself in a host — the obligate biochemical the most important in symbiotic systems; transport, interdependence between two endosymbionts can evo- indicating that the bacteriocyte mediates exchange lutionarily seal the bacterial metabolic complementa- of metabolites and substrates; and production of lys- tion, and the establishment of a stable consortium would ozyme, possibly involved in nutrient uptake by the be the expected evolutionary outcome. This appears host, as already mentioned. A similar study that was to be the case in the sharpshooter H. coagulata, with carried out in S. zeamais revealed that the bacteriocyte two complementary endosymbionts, B. cicadellinicola also allows increased sugar uptake and metabolism, and and S. muelleri15,23. Whereas B. cicadellinicola is mainly various anti-stress systems that control the resultant devoted to the biosynthesis of vitamins, S. muelleri oxidative stress75. encodes the enzymes that are involved in the biosynthesis of most essential amino acids. The complementarity Evolutionary outcomes of symbioses is striking. For example, S. muelleri has lost the pathway Once an association has been established and the for the biosynthesis of histidine, which is the only bio- genome-reduction process has started, the loss of some synthetic pathway for essential amino acids retained in metabolic abilities, and other functions, irreversibly ties B. cicadellinicola.

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Box 2 | Minimal cells and synthetic biology elucidated whether C. ruddii is on its way to becoming an organelle, or if it is nearing extinction and replace- The phenomenon of genome downsizing that has been observed in endosymbionts, ment by an unidentified symbiont61. The transfer of intracellular parasites and organelles has inspired a research programme on minimal genes from an organelle to the nucleus has been demon- life, based on the hypothesis that genomes must retain essential genes that are ref. 83 involved in housekeeping functions, and a minimum number of metabolic strated under laboratory conditions (see and ref- transactions for cellular survival and replication. Apart from shedding light on this erences therein). There is also evidence of gene transfer fundamental topic, the search for minimal genomes is of much value in the context from Wolbachia to the nuclear genome of several insect of synthetic biology. In fact, one of the aims of this emerging field is the definition and nematode hosts84. These remarkable cases of lateral and chemical synthesis of a minimal genome, and its incorporation and expression gene transfer should be a caution for those carrying out in a suitable chassis, either derived from a cell (top-down strategy) or starting from a eukaryotic genome sequencing, and should add new simple chemical system, such as a liposome (bottom-up approach). The top-down insights of relevance to the debate about the differences 107 approach, also called genome-driven cell engineering or the minimal cell between cell organelles and endosymbionts85. project108, is expected to provide methods for fabricating engineered cells, which will have a big impact both on biotechnology and on our basic understanding of Conclusions and future directions living systems109. The first attempt to define a minimal genome based on comparative genomics used The advent of genomics has significantly contributed the genomes of two human parasitic bacteria: Haemophilus influenzae and to reinforcing the view of symbiosis as a widespread Mycoplasma genitalium110. Owing to their parasitic lifestyles, these two bacteria have biological phenomenon, especially through the genome reduced genomes when compared with their closest free-living relatives. The sequencing of uncultivable organisms. Symbiosis is a analysis led to the proposal of a minimal gene set composed of just 256 genes, most dynamic process in which the prokaryotic symbiont, of them involved in genetic-information storage and processing, protein while providing the host with new metabolic capa- chaperoning and a limited metabolic capability. Later, a combined study of all bilities, experiences many genotypic and phenotypic published research using computational or experimental methods, including the changes compared with its free-living relatives in order comparison of reduced genomes from insect endosymbionts, was used to define to adapt to its new lifestyle, as revealed by the specific the minimal core of essential genes for a free-living bacterium thriving in a differences observed in the completely sequenced chemically rich environment111. The main difference between this study and previous efforts (see ref. 112 and references therein) was the emphasis on the functional genomes of several endosymbiotic bacteria. completeness of the minimal metabolism encoded by the proposed gene repertoire Many questions remain open for future research. On (involving 62 protein-coding genes out of a minimal set of 208 genes). This aspect has the host side, a better understanding of how these organ- been explored further, demonstrating the stoichiometric consistency of the minimal isms control and/or domesticate prokaryotic symbionts metabolic network, as well as providing insights into some of its architectural is needed. Genomic and transcriptomic studies of model properties, such as its size, clustering and robustness113. eukaryotic hosts that have well known and stable sym- It must be stressed that these latter studies present just one possible form of a biotic associations will be valuable. Until now, only the minimal metabolism. Metabolic complexity is ecologically dependent; it is a genome of B. malayi, the nematode host of W. pipiensis function of the chemical richness of the environment and the primary energy wBm, has been fully sequenced78, but the sequence of the source(s) available to the living system (FIG. 1). Different versions of minimal gene aphid A. pisum is on its way. Comparative analysis with sets exist, that is, different combinations of metabolic pathways that can perform the essential biological functions of self-maintenance, self-reproduction and model organisms that have already been sequenced, 86 evolution114 under the same external conditions. However, it is possible to define such as the nematode Caenorhabditis elegans and the 87 which functions should be performed in any living cell in a specified niche, and list arthropod D. melanogaster , will give new insights the genes that would be necessary to maintain such functions. In this context, into the changes that are experienced by the host after comparative and evolutionary genomics of endosymbionts provide insights into the the establishment of the symbiosis. On the bacterial different routes that endosymbionts have taken to solve the challenges of their own side, comparative studies of the molecular processes maintenance under the diverse selective pressures and environmental constraints governing the nature of symbiotic associations, either that are the result of different host lifestyles. mutualistic or parasitic, will provide important clues to understand why microorganisms evolve toward one lifestyle or the other. The genome degeneration process seen in endosymbi- Symbiosis is not always a matter of two organisms: onts can also be accompanied by massive gene transfer to we are gaining increasing evidence that well-established the host cell nucleus82. In order for a true cell organelle prokaryotic endosymbionts also maintain some meta- to be established, several processes must be acquired: new bolic interplay with new facultative symbionts. Such gene-regulatory mechanisms; specific target sequences relationships could finish with the extinction of the for proteins that are encoded by the host nucleus but P‑endosymbiont (as described in insects from the fam- function in the symbiont; and a protein import appa- ily Dryopthoridae, to which Sitophilus spp. belongs56, or ratus that functions at the symbiont surface. These the Buchnera–Serratia consortium in cedar aphids21), processes occurred in the α‑proteobacterial ancestor or the symbiotic consortia could continue over time of mitochondria and in the cyanobacterial ances- (as described in the Baumannia–Sulcia consortium in 15 Minimal genome tor that gave rise to chloroplast lineages. Is there any sharpshooters ). Metagenomics and systems biology The smallest set of genes that system that is currently en route from endosymbiont are new approaches that can be used in the analysis is necessary and sufficient to to organelle? At present, the smallest sequenced endo- of complex symbiotic consortia. For example, the sustain a living cell in the most symbiont genome belongs to C. ruddii, which only metagenomes of sponge microbial communities have favourable conditions; that is, in the presence of adequate codes for approximately 180 proteins. However, a care- been shown to contain genes and gene clusters for the nutrients and in the absence ful analysis revealed that many genes that are involved biosynthesis of biologically active natural products. of stress factors. in essential functions are absent. It remains to be There is evidence to suggest that a mutually beneficial

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Synthetic biology relationship exists, at least between some of the bacte- new insights into symbiotic associations, including 88 The design and fabrication of ria and the sponges themselves . Another interesting those that are highly relevant to biotechnology and artificial biological systems, example is the symbiotic gut microbiome of mammals, biomedicine. with the aim of either in which we are starting to have a clearer picture of the Finally, synthetic biology will benefit from the new optimizing their performance in the context of their role of the symbiotic microbial consortia in the meta- insights gained by comparative genomic analyses 89 technological utility or of bolic phenotype of the mammalian host . In this case, of extremely reduced genomes (BOX 2). This type of deepening our understanding an extensive microbial–mammalian co-metabolism research makes important contributions to the concep- of the naturally occurring exists. As new complete genomes of eukaryote hosts tually intriguing and biologically fundamental question organisms. become available, we will be able to make new meta- of the minimal genomic basis for sustaining cellular life. bolic inferences to understand the interplay between Many efforts in this area have attracted a great deal of the host and its associated microbiota. Genomics and public attention regarding both biosecurity risks and metagenomics are becoming essential tools to provide potential commercialization.

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