Tracing the evolution of gene loss in obligate bacterial symbionts

The gamma-proteobacterial symbionts of insects are a model group for comparative studies of genome reduction. The phylogenetic proximity of these reduced genomes to the larger genomes of well-studied free-living bacteria has enabled reconstructions of the process by which genes and DNA are lost. Three genome sequences are now available for Buchnera aphidicola. Analyses of Buchnera genomes in comparison with those of related enteric bacteria suggest that extensive changes including large deletions, repetitive element proliferation and chromosomal rearrangements occurred initially, followed by extreme stasis in gene order and slow decay of additional genes. This pattern appears to be characteristic of symbiont evolution.     

Genomic signatures of obligate host dependence in the luminous bacterial symbiont of a vertebrate

The majority of bacteria engaged in bioluminescent symbiosis are environmentally acquired and facultatively symbiotic. A few enigmatic bioluminescent symbionts have not been successfully cultured, which has led to speculation that they may be obligately dependent on their hosts. Here, we report the draft genome of the uncultured luminous symbiont of an anomalopid flashlight fish, ‘Candidatus Photodesmus katoptron’. The genome of the anomalopid symbiont is reduced by 80% compared with close relatives and lacks almost all genes necessary for amino acid synthesis and for metabolism of energy sources other than glucose, supporting obligate dependence on the host for growth. ‘Candidatus Photodesmus katoptron’ is the first described obligate mutualistic symbiont of a vertebrate. Unlike most other obligate mutualists, the anomalopid symbiont genome has retained complete pathways for chemotaxis and motility as well as most genes involved in cell wall production, consistent with the hypothesis that these bacteria may be transmitted environmentally during an extra‐host phase.     

Transitional genomes and nutritional role reversals identified for dual symbionts of adelgids (Aphidoidea: Adelgidae)

Many plant-sap-feeding insects have maintained a single, obligate, nutritional symbiont over the long history of their lineage. This senior symbiont may be joined by one or more junior symbionts that compensate for gaps in function incurred through genome-degradative forces. Adelgids are sap-sucking insects that feed solely on conifer trees and follow complex life cycles in which the diet fluctuates in nutrient levels. Adelgids are unusual in that both senior and junior symbionts appear to have been replaced repeatedly over their evolutionary history. Genomes can provide clues to understanding symbiont replacements, but only the dual symbionts of hemlock adelgids have been examined thus far. Here, we sequence and compare genomes of four additional dual-symbiont pairs in adelgids. We show that these symbionts are nutritional partners originating from diverse bacterial lineages and exhibiting wide variation in general genome characteristics. Although dual symbionts cooperate to produce nutrients, the balance of contributions varies widely across pairs, and total genome contents reflect a range of ages and degrees of degradation. Most symbionts appear to be in transitional states of genome reduction. Our findings support a hypothesis of periodic symbiont turnover driven by fluctuating selection for nutritional provisioning related to gains and losses of complex life cycles in their hosts.Subject terms: Microbial ecology, Evolution, Genomics     

Independent origins and horizontal transfer of bacterial symbionts of aphids

Many insect groups have obligate associations with primary endosymbionts: mutualistic bacteria that are maternally transmitted and derived from an ancient infection. Often, the same insects are hosts to 'secondary' bacterial symbionts which are maternally transmitted but relatively labile within host lineages. To explore the dynamics of secondary symbiont associations in aphids, we characterized bacteria infecting 15 species of macrosiphine aphids using DNA sequencing, diagnostic polymerase chain reaction (PCR), diagnostic restriction digests, phylogenetic analyses, and electron microscopy to examine aphids from nature and from laboratory colonies. Three types of bacteria besides Buchnera were found repeatedly; all three fall within the Enterobacteriaceae. The R-type has a 16S rDNA less than 0.1% different from that of the secondary symbiont previously reported from Acyrthosiphon pisum and is related to Serratia species. The T-type includes a symbiont previously reported from a whitefly; the U-type comprises a new cluster near the T-type. The T-type was found in every one of 40 Uroleucon ambrosiae clones collected throughout the United States. In contrast, A. pisum individuals were infected by any combination of the three symbiont types. Secondary symbionts were maternally transmitted for 11 months within laboratory-reared A. pisum clones and were present in sexually produced eggs. PCR screens for a bacteriophage, APSE-1, indicated its presence in both A. pisum and U. ambrosiae containing secondary symbionts. Electron microscopy of R-type and T-type bacteria in A. pisum and in U. ambrosiae revealed rod-shaped organisms that attain extremely high densities within a few bacteriocytes.     

Swapping symbionts in spittlebugs: evolutionary replacement of a reduced genome symbiont

Bacterial symbionts that undergo long-term maternal transmission experience elevated fixation of deleterious mutations, resulting in massive loss of genes and changes in gene sequences that appear to limit efficiency of gene products. Potentially, this dwindling of symbiont functionality impacts hosts that depend on these bacteria for nutrition. One evolutionary escape route is the acquisition of a novel symbiont with a robust genome and metabolic capabilities. Such an acquisition has occurred in an ancestor of Philaenus spumarius, the meadow spittlebug (Insecta: Cercopoidea), which has replaced its ancient association with the tiny genome symbiont Zinderia insecticola (Betaproteobacteria) with an association with a symbiont related to Sodalis glossinidius (Gammaproteobacteria). Spittlebugs feed exclusively on xylem sap, a diet that is low both in essential amino acids and in sugar or other substrates for energy production. The new symbiont genome has undergone proliferation of mobile elements resulting in many gene inactivations; nonetheless, it has selectively maintained genes replacing functions of its predecessor for amino-acid biosynthesis. Whereas ancient symbiont partners typically retain perfectly complementary sets of amino-acid biosynthetic pathways, the novel symbiont introduces some redundancy as it retains some pathways also present in the partner symbionts (Sulcia muelleri). Strikingly, the newly acquired Sodalis-like symbiont retains genes underlying efficient routes of energy production, including a complete TCA cycle, potentially relaxing the severe energy limitations of the xylem-feeding hosts. Although evolutionary replacements of ancient symbionts are infrequent, they potentially enable evolutionary and ecological novelty by conferring novel metabolic capabilities to host lineages.     

Microbial minimalism: genome reduction in bacterial pathogens

When bacterial lineages make the transition from free-living or facultatively parasitic life cycles to permanent associations with hosts, they undergo a major loss of genes and DNA. Complete genome sequences are providing an understanding of how extreme genome reduction affects evolutionary directions and metabolic capabilities of obligate pathogens and symbionts.     

Bacterial Endosymbiosis in a Chordate Host: Long-Term Co-Evolution and Conservation of Secondary Metabolism

Intracellular symbiosis is known to be widespread in insects, but there are few described examples in other types of host. These symbionts carry out useful activities such as synthesizing nutrients and conferring resistance against adverse events such as parasitism. Such symbionts persist through host speciation events, being passed down through vertical transmission. Due to various evolutionary forces, symbionts go through a process of genome reduction, eventually resulting in tiny genomes where only those genes essential to immediate survival and those beneficial to the host remain. In the marine environment, invertebrates such as tunicates are known to harbor complex microbiomes implicated in the production of natural products that are toxic and probably serve a defensive function. Here, we show that the intracellular symbiont Candidatus Endolissoclinum faulkneri is a long-standing symbiont of the tunicate Lissoclinum patella, that has persisted through cryptic speciation of the host. In contrast to the known examples of insect symbionts, which tend to be either relatively recent or ancient relationships, the genome of Ca. E. faulkneri has a very low coding density but very few recognizable pseudogenes. The almost complete degradation of intergenic regions and stable gene inventory of extant strains of Ca. E. faulkneri show that further degradation and deletion is happening very slowly. This is a novel stage of genome reduction and provides insight into how tiny genomes are formed. The ptz pathway, which produces the defensive patellazoles, is shown to date to before the divergence of Ca. E. faulkneri strains, reinforcing its importance in this symbiotic relationship. Lastly, as in insects we show that stable symbionts can be lost, as we describe an L. patella animal where Ca. E. faulkneri is displaced by a likely intracellular pathogen. Our results suggest that intracellular symbionts may be an important source of ecologically significant natural products in animals.     

Exploration of the core metabolism of symbiotic bacteria

ABSTRACT: BACKGROUND: A large number of genome-scale metabolic networks is now available for many organisms, mostly bacteria. Previous works on minimal gene sets, when analysing host-dependent bacteria, found small common sets of metabolic genes. When such analyses are restricted to bacteria with similar lifestyles, larger portions of metabolism are expected to be shared and their composition is worth investigating. Here we report a comparative analysis of the small molecule metabolism of symbiotic bacteria, exploring common and variable portions as well as the contribution of different lifestyle groups to the reduction of a common set of metabolic capabilities. RESULTS: We found no reaction shared by all the bacteria analysed. Disregarding those with the smallest genomes, we still do not find a reaction core, however we did find a core of biochemical capabilities. While obligate intracellular symbionts have no core of reactions within their group, extracellular and cell-associated symbionts do have a small core composed of disconnected fragments. In agreement with previous findings in Escherichia coli, their cores are enriched in biosynthetic processes whereas the variable metabolisms have similar ratios of biosynthetic and degradation reactions. Conversely, the variable metabolism of obligate intracellular symbionts is enriched in anabolism. CONCLUSION: Even when removing the symbionts with the most reduced genomes, there is no core of reactions common to the analysed symbiotic bacteria. The main reason is the very high specialisation of obligate intracellular symbionts, however, host-dependence alone is not an explanation for such absence. The composition of the metabolism of cell-associated and extracellular bacteria shows that while they have similar needs in terms of the building blocks of their cells, they have to adapt to very distinct environments. On the other hand, in obligate intracellular bacteria, catabolism has largely disappeared, whereas synthetic routes appear to have been selected for depending on the nature of the symbiosis. As more genomes are added, we expect, based on our simulations, that the core of cell-associated and extracellular bacteria continues to diminish, converging to approximately 60 reactions.     

Reduced genome of the thioautotrophic intracellular symbiont in a deep-sea clam, Calyptogena okutanii

Although dense animal communities at hydrothermal vents and cold seeps rely on symbioses with chemoautotrophic bacteria [1, 2], knowledge of the mechanisms underlying these chemosynthetic symbioses is still fragmentary because of the difficulty in culturing the symbionts and the hosts in the laboratory. Deep-sea Calyptogena clams harbor thioautotrophic bacterial symbionts in their gill epithelial cells [1, 2]. They have vestigial digestive tracts and nutritionally depend on their symbionts [3], which are vertically transmitted via eggs [4]. To clarify the symbionts' metabolic roles in the symbiosis and adaptations to intracellular conditions, we present the complete genome sequence of the symbiont of Calyptogena okutanii. The genome is a circular chromosome of 1,022,154 bp with 31.6% guanine + cytosine (G + C) content, and is the smallest reported genome in autotrophic bacteria. It encodes 939 protein-coding genes, including those for thioautotrophy and for the syntheses of almost all amino acids and various cofactors. However, transporters for these substances to the host cell are apparently absent. Genes that are unnecessary for an intracellular lifestyle, as well as some essential genes (e.g., ftsZ for cytokinesis), appear to have been lost from the symbiont genome. Reductive evolution of the genome might be ongoing in the vertically transmitted Calyptogena symbionts.     

Genome evolution in bacterial endosymbionts of insects

Many insect species rely on intracellular bacterial symbionts for their viability and fecundity. Large-scale DNA-sequence analyses are revealing the forces that shape the evolution of these bacterial associates and the genetic basis of their specialization to an intracellular lifestyle. The full genome sequences of two obligate mutualists, Buchnera aphidicola of aphids and Wigglesworthia glossinidia of tsetse flies, reveal substantial gene loss and an integration of host and symbiont metabolic functions. Further genomic comparisons should reveal the generality of these features among bacterial mutualists and the extent to which they are shared with other intracellular bacteria, including obligate pathogens.     

Correlations Between Bacterial Ecology and Mobile DNA

Several factors can affect the density of mobile DNA in bacterial genomes including rates of exposure to novel gene pools, recombination, and reductive evolution. These traits are difficult to measure across a broad range of bacterial species, but the ecological niches occupied by an organism provide some indication of the relative magnitude of these forces. Here, by analyzing 384 bacterial genomes assigned to three ecological categories (obligate intracellular, facultative intracellular, and extracellular), we address two, related questions: How does the density of mobile DNA vary across the Bacteria? And is there a statistically supported relationship between ecological niche and mobile element gene density? We report three findings. First, the fraction of mobile element genes in bacterial genomes ranges from 0 to 21% and decreases significantly: facultative intracellular > extracellular > obligate intracellular bacteria. Results further show that the obligate intracellular bacteria that host switch have a higher mobile DNA gene density than the obligate intracellular bacteria that are vertically transmitted. Second, while bacteria from the three ecological niches differ in their average mobile DNA contents, the ranges of mobile DNA found in each category overlap a surprising extent, suggesting bacteria with different lifestyles can tolerate similar amounts of mobile DNA. Third, mobile DNA gene densities increase with genome size across the entire dataset, and the significance of this correlation is dependent on the obligate intracellular bacteria. Further, mobile DNA gene densities do not correlate with evolutionary relationships in a 16S rDNA phylogeny. These findings statistically support a compelling link between mobile element evolution and bacterial ecology.     

Intracellular symbionts of sharpshooters (Insecta: Hemiptera: Cicadellinae) form a distinct clade with a small genome

The leafhoppers (Insecta: Hemiptera: Cicadellidae) are the most species-rich group of invertebrates in which intracellular symbionts are usual. Here we present the first molecular characterization of bacteriome-associates in the leafhoppers, with focus on the subfamily Cicadellinae (sharpshooters). Phylogenetic analyses of 16S rDNA sequences from intracellular symbionts residing in the bacteriomes of five host species indicate that these symbionts form a well-defined clade within the gamma-3 Proteobacteria, consistent with an ancient colonization and strict vertical transmission. More extensive gene sequence information is reported for the symbiont of Homalodisca coagulata (Say). The genome size, as determined by pulsed field gel electrophoresis, is approximately 680 kb. This finding, when combined with published results for symbionts of aphids, ants, psyllids and tsetse flies, adds to an emerging pattern which suggests that bacteriome associates often descend from ancient infections by gamma Proteobacteria, and that these lineages have undergone pronounced genome reduction. A new genus and species name, 'Candidatus Baumannia cicadellinicola' (sp. nov.) is proposed for this newly characterized clade of symbiotic bacteria.     

Comparative genomics of Pneumocystis carinii with other protists: implications for life style

Three protistan genomes were analyzed for differential genetic traits that may be associated with biological adaptations to their unique life styles. The microsporidian, Encephalitozoon cuniculi, an obligate intracellular parasite; the ascomycetes, Pneumocystis carinii, considered an opportunistic pathogen; and Saccharomyces cerevisiae, a model organism exhibiting a free-living life style, were used in comparisons of genomic architecture, reproductive strategies, and metabolic capacity predicted by the presence of signature genes. Genome size, gene number, and metabolic function decreased as the organisms became more dependent on their hosts. In contrast, gene density and the percentage of genes dedicated to cell growth and division were substantially increased in the genome of E. cuniculi. The obligate life style was associated with reductions in gene number, genome size, and reduced metabolic capacity while the free-living life style was coincident with gene duplications and duplication of large portions of the genome. The genomic characteristics and metabolic capacity of P. carinii were usually intermediate between those of the other two protistan genomes, but unique characteristics such as the presence of a single rDNA locus may indicate that these organisms could be in the process of becoming more host dependent.     

Nutrient provisioning facilitates homeostasis between tsetse fly (Diptera: Glossinidae) symbionts

Host-associated microbial interactions may involve genome complementation, driving-enhanced communal efficiency and stability. The tsetse fly (Diptera: Glossinidae), the obligate vector of African trypanosomes (Trypanosoma brucei subspp.), harbours two enteric Gammaproteobacteria symbionts: Wigglesworthia glossinidia and Sodalis glossinidius. Host coevolution has streamlined the Wigglesworthia genome to complement the exclusively sanguivorous tsetse lifestyle. Comparative genomics reveal that the Sodalis genome contains the majority of Wigglesworthia genes. This significant genomic overlap calls into question why tsetse maintains the coresidence of both symbionts and, furthermore, how symbiont homeostasis is maintained. One of the few distinctions between the Wigglesworthia and Sodalis genomes lies in thiamine biosynthesis. While Wigglesworthia can synthesize thiamine, Sodalis lacks this capability but retains a thiamine ABC transporter (tbpAthiPQ) believed to salvage thiamine. This genetic complementation may represent the early convergence of metabolic pathways that may act to retain Wigglesworthia and evade species antagonism. We show that thiamine monophosphate, the specific thiamine derivative putatively synthesized by Wigglesworthia, impacts Sodalis thiamine transporter expression, proliferation and intracellular localization. A greater understanding of tsetse symbiont interactions may generate alternative control strategies for this significant medical and agricultural pest, while also providing insight into the evolution of microbial associations within hosts.     

Symbiosis and insect diversification: an ancient symbiont of sap-feeding insects from the bacterial phylum Bacteroidetes

Several insect groups have obligate, vertically transmitted bacterial symbionts that provision hosts with nutrients that are limiting in the diet. Some of these bacteria have been shown to descend from ancient infections. Here we show that the large group of related insects including cicadas, leafhoppers, treehoppers, spittlebugs, and planthoppers host a distinct clade of bacterial symbionts. This newly described symbiont lineage belongs to the phylum Bacteroidetes. Analyses of 16S rRNA genes indicate that the symbiont phylogeny is completely congruent with the phylogeny of insect hosts as currently known. These results support the ancient acquisition of a symbiont by a shared ancestor of these insects, dating the original infection to at least 260 million years ago. As visualized in a species of spittlebug (Cercopoidea) and in a species of sharpshooter (Cicadellinae), the symbionts have extraordinarily large cells with an elongate shape, often more than 30 mum in length; in situ hybridizations verify that these correspond to the phylum Bacteroidetes. "Candidatus Sulcia muelleri" is proposed as the name of the new symbiont.     

Genomes at the interface between bacteria and organelles

The topic of the transition of the genome of a free-living bacterial organism to that of an organelle is addressed by considering three cases. Two of these are relatively clear-cut as involving respectively organisms (cyanobacteria) and organelles (plastids). Cyanobacteria are usually free-living but some are involved in symbioses with a range of eukaryotes in which the cyanobacterial partner contributes photosynthesis, nitrogen fixation, or both of these. In several of these symbioses the cyanobacterium is vertically transmitted, and in a few instances, sufficient unsuccessful attempts have been made to culture the cyanobiont independently for the association to be considered obligate for the cyanobacterium. Plastids clearly had a cyanobacterial ancestor but cannot grow independently of the host eukaryote. Plastid genomes have at most 15% of the number of genes encoded by the cyanobacterium with the smallest number of genes; more genes than are retained in the plastid genome have been transferred to the eukaryote nuclear genome, while the rest of the cyanobacterial genes have been lost. Even the most cyanobacteria-like plastids, for example the "cyanelles" of glaucocystophyte algae, are functionally and genetically very similar to other plastids and give little help in indicating intermediates in the evolution of plastids. The third case considered is the vertically transmitted intracellular bacterial symbionts of insects where the symbiosis is usually obligate for both partners. The number of genes encoded by the genomes of these obligate symbionts is intermediate between that of organelles and that of free-living bacteria, and the genomes of the insect symbionts also show rapid rates of sequence evolution and AT (adenine, thymine) bias. Genetically and functionally, these insect symbionts show considerable similarity to organelles.     

Loss of genes for DNA recombination and repair in the reductive genome evolution of thioautotrophic symbionts of Calyptogena clams

Background

Two Calyptogena clam intracellular obligate symbionts, Ca. Vesicomyosocius okutanii (Vok; C. okutanii symbiont) and Ca. Ruthia magnifica (Rma; C. magnifica symbiont), have small genomes (1.02 and 1.16 Mb, respectively) with low G+C contents (31.6% and 34.0%, respectively) and are thought to be in an ongoing stage of reductive genome evolution (RGE). They lack recA and some genes for DNA repair, including mutY. The loss of recA and mutY is thought to contribute to the stabilization of their genome architectures and GC bias, respectively. To understand how these genes were lost from the symbiont genomes, we surveyed these genes in the genomes from 10 other Calyptogena clam symbionts using the polymerase chain reaction (PCR).

Results

Phylogenetic trees reconstructed using concatenated 16S and 23S rRNA gene sequences showed that the symbionts formed two clades, clade I (symbionts of C. kawamurai, C. laubieri, C. kilmeri, C. okutanii and C. soyoae) and clade II (those of C. pacifica, C. fausta, C. nautilei, C. stearnsii, C. magnifica, C. fossajaponica and C. phaseoliformis). recA was detected by PCR with consensus primers for recA in the symbiont of C. phaseoliformis. A detailed homology search revealed a remnant recA in the Rma genome. Using PCR with a newly designed primer set, intact recA or its remnant was detected in clade II symbionts. In clade I symbionts, the recA coding region was found to be mostly deleted. In the Rma genome, a pseudogene of mutY was found. Using PCR with newly designed primer sets, mutY was not found in clade I symbionts but was found in clade II symbionts. The G+C content of 16S and 23S rRNA genes in symbionts lacking mutY was significantly lower than in those with mutY.

Conclusions

The extant Calyptogena clam symbionts in clade II were shown to have recA and mutY or their remnants, while those in clade I did not. The present results indicate that the extant symbionts are losing these genes in RGE, and that the loss of mutY contributed to the GC bias of the genomes during their evolution.