The Genomics and Cell Biology of Host-Beneficial Intracellular Infections.

Microbes gain access to eukaryotic cells as food for bacteria-grazing protists, for host protection by microbe-killing immune cells, or for microbial benefit when pathogens enter host cells to replicate. But microbes can also gain access to a host cell and become an important-often required-beneficial partner. The oldest beneficial microbial infections are the ancient eukaryotic organelles now called the mitochondrion and plastid. But numerous other host-beneficial intracellular infections occur throughout eukaryotes. Here I review the genomics and cell biology of these interactions with a focus on intracellular bacteria. The genomes of host-beneficial intracellular bacteria have features that span a previously unfilled gap between pathogens and organelles. Host cell adaptations to allow the intracellular persistence of beneficial bacteria are found along with evidence for the microbial manipulation of host cells, but the cellular mechanisms of beneficial bacterial infections are not well understood.

Peptidoglycan Production by an Insect-Bacterial Mosaic.

Peptidoglycan (PG) is a defining feature of bacteria, involved in cell division, shape, and integrity. We previously reported that several genes related to PG biosynthesis were horizontally transferred from bacteria to the nuclear genome of mealybugs. Mealybugs are notable for containing a nested bacteria-within-bacterium endosymbiotic structure in specialized insect cells, where one bacterium, Moranella, lives in the cytoplasm of another bacterium, Tremblaya. Here we show that horizontally transferred genes on the mealybug genome work together with genes retained on the Moranella genome to produce a PG layer exclusively at the Moranella cell periphery. Furthermore, we show that an insect protein encoded by a horizontally transferred gene of bacterial origin is transported into the Moranella cytoplasm. These results provide a striking parallel to the genetic and biochemical mosaicism found in organelles, and prove that multiple horizontally transferred genes can become integrated into a functional pathway distributed between animal and bacterial endosymbiont genomes.

Sympatric speciation in a bacterial endosymbiont results in two genomes with the functionality of one.

Mutualisms that become evolutionarily stable give rise to organismal interdependencies. Some insects have developed intracellular associations with communities of bacteria, where the interdependencies are manifest in patterns of complementary gene loss and retention among members of the symbiosis. Here, using comparative genomics and microscopy, we show that a three-member symbiotic community has become a four-way assemblage through a novel bacterial lineage-splitting event. In some but not all cicada species of the genus Tettigades, the endosymbiont Candidatus Hodgkinia cicadicola has split into two new cytologically distinct but metabolically interdependent species. Although these new bacterial genomes are partitioned into discrete cell types, the intergenome patterns of gene loss and retention are almost perfectly complementary. These results defy easy classification: they show genomic patterns consistent with those observed after both speciation and whole-genome duplication. We suggest that our results highlight the potential power of nonadaptive forces in shaping organismal complexity.

Horizontal gene transfer from diverse bacteria to an insect genome enables a tripartite nested mealybug symbiosis.

The smallest reported bacterial genome belongs to Tremblaya princeps, a symbiont of Planococcus citri mealybugs (PCIT). Tremblaya PCIT not only has a 139 kb genome, but possesses its own bacterial endosymbiont, Moranella endobia. Genome and transcriptome sequencing, including genome sequencing from a Tremblaya lineage lacking intracellular bacteria, reveals that the extreme genomic degeneracy of Tremblaya PCIT likely resulted from acquiring Moranella as an endosymbiont. In addition, at least 22 expressed horizontally transferred genes from multiple diverse bacteria to the mealybug genome likely complement missing symbiont genes. However, none of these horizontally transferred genes are from Tremblaya, showing that genome reduction in this symbiont has not been enabled by gene transfer to the host nucleus. Our results thus indicate that the functioning of this three-way symbiosis is dependent on genes from at least six lineages of organisms and reveal a path to intimate endosymbiosis distinct from that followed by organelles.

How do bacterial endosymbionts work with so few genes?

The move from a free-living environment to a long-term residence inside a host eukaryotic cell has profound effects on bacterial function. While endosymbioses are found in many eukaryotes, from protists to plants to animals, the bacteria that form these host-beneficial relationships are even more diverse. Endosymbiont genomes can become radically smaller than their free-living relatives, and their few remaining genes show extreme compositional biases. The details of how these reduced and divergent gene sets work, and how they interact with their host cell, remain mysterious. This Unsolved Mystery reviews how genome reduction alters endosymbiont biology and highlights a "tipping point" where the loss of the ability to build a cell envelope coincides with a marked erosion of translation-related genes.

Pseudofinder: Detection of Pseudogenes in Prokaryotic Genomes.

Prokaryotic genomes are usually densely packed with intact and functional genes. However, in certain contexts, such as after recent ecological shifts or extreme population bottlenecks, broken and nonfunctional gene fragments can quickly accumulate and form a substantial fraction of the genome. Identification of these broken genes, called pseudogenes, is a critical step for understanding the evolutionary forces acting upon, and the functional potential encoded within, prokaryotic genomes. Here, we present Pseudofinder, an open-source software dedicated to pseudogene identification and analysis in bacterial and archaeal genomes. We demonstrate that Pseudofinder's multi-pronged, reference-based approach can detect a wide variety of pseudogenes, including those that are highly degraded and typically missed by gene-calling pipelines, as well newly formed pseudogenes containing only one or a few inactivating mutations. Additionally, Pseudofinder can detect genes that lack inactivating substitutions but experiencing relaxed selection. Implementation of Pseudofinder in annotation pipelines will allow more precise estimations of the functional potential of sequenced microbes, while also generating new hypotheses related to the evolutionary dynamics of bacterial and archaeal genomes.

Lichen fungi do not depend on the alga for ATP production: A comment on Pogoda et al. (2018).

Lichen fungi live in a symbiotic association with unicellular phototrophs and most have no known aposymbiotic stage. A recent study in Molecular Ecology postulated that some of them have lost mitochondrial oxidative phosphorylation and rely on their algal partners for ATP. This claim originated from an apparent lack of ATP9, a gene encoding one subunit of ATP synthase, from a few mitochondrial genomes. Here, we show that while these fungi indeed have lost the mitochondrial ATP9, each retain a nuclear copy of this gene. Our analysis reaffirms that lichen fungi produce their own ATP.

Recurrent symbiont recruitment from fungal parasites in cicadas.

Diverse insects are associated with ancient bacterial symbionts, whose genomes have often suffered drastic reduction and degeneration. In extreme cases, such symbiont genomes seem almost unable to sustain the basic cellular functioning, which comprises an open question in the evolution of symbiosis. Here, we report an insect group wherein an ancient symbiont lineage suffering massive genome erosion has experienced recurrent extinction and replacement by host-associated pathogenic microbes. Cicadas are associated with the ancient bacterial co-obligate symbionts Sulcia and Hodgkinia, whose streamlined genomes are specialized for synthesizing essential amino acids, thereby enabling the host to live on plant sap. However, our inspection of 24 Japanese cicada species revealed that while all species possessed Sulcia, only nine species retained Hodgkinia, and their genomes exhibited substantial structural instability. The remaining 15 species lacked Hodgkinia and instead harbored yeast-like fungal symbionts. Detailed phylogenetic analyses uncovered repeated Hodgkinia-fungus and fungus-fungus replacements in cicadas. The fungal symbionts were phylogenetically intermingled with cicada-parasitizing Ophiocordyceps fungi, identifying entomopathogenic origins of the fungal symbionts. Most fungal symbionts of cicadas were uncultivable, but the fungal symbiont of Meimuna opalifera was cultivable, possibly because it is at an early stage of fungal symbiont replacement. Genome sequencing of the fungal symbiont revealed its metabolic versatility, presumably capable of synthesizing almost all amino acids, vitamins, and other metabolites, which is more than sufficient to compensate for the Hodgkinia loss. These findings highlight a straightforward ecological and evolutionary connection between parasitism and symbiosis, which may provide an evolutionary trajectory to renovate deteriorated ancient symbiosis via pathogen domestication.

Multiple origins of interdependent endosymbiotic complexes in a genus of cicadas.

Bacterial endosymbionts that provide nutrients to hosts often have genomes that are extremely stable in structure and gene content. In contrast, the genome of the endosymbiont Hodgkinia cicadicola has fractured into multiple distinct lineages in some species of the cicada genus Tettigades To better understand the frequency, timing, and outcomes of Hodgkinia lineage splitting throughout this cicada genus, we sampled cicadas over three field seasons in Chile and performed genomics and microscopy on representative samples. We found that a single ancestral Hodgkinia lineage has split at least six independent times in Tettigades over the last 4 million years, resulting in complexes of between two and six distinct Hodgkinia lineages per host. Individual genomes in these symbiotic complexes differ dramatically in relative abundance, genome size, organization, and gene content. Each Hodgkinia lineage retains a small set of core genes involved in genetic information processing, but the high level of gene loss experienced by all genomes suggests that extensive sharing of gene products among symbiont cells must occur. In total, Hodgkinia complexes that consist of multiple lineages encode nearly complete sets of genes present on the ancestral single lineage and presumably perform the same functions as symbionts that have not undergone splitting. However, differences in the timing of the splits, along with dissimilar gene loss patterns on the resulting genomes, have led to very different outcomes of lineage splitting in extant cicadas.

One Hundred Mitochondrial Genomes of Cicadas.

Mitochondrial genomes can provide valuable information on the biology and evolutionary histories of their host organisms. Here, we present and characterize the complete coding regions of 107 mitochondrial genomes (mitogenomes) of cicadas (Insecta: Hemiptera: Auchenorrhyncha: Cicadoidea), representing 31 genera, 61 species, and 83 populations. We show that all cicada mitogenomes retain the organization and gene contents thought to be ancestral in insects, with some variability among cicada clades in the length of a region between the genes nad2 and cox1, which encodes 3 tRNAs. Phylogenetic analyses using these mitogenomes recapitulate a recent 5-gene classification of cicadas into families and subfamilies, but also identify a species that falls outside of the established taxonomic framework. While protein-coding genes are under strong purifying selection, tests of relative evolutionary rates reveal significant variation in evolutionary rates across taxa, highlighting the dynamic nature of mitochondrial genome evolution in cicadas. These data will serve as a useful reference for future research into the systematics, ecology, and evolution of the superfamily Cicadoidea.

Repeated replacement of an intrabacterial symbiont in the tripartite nested mealybug symbiosis.

Stable endosymbiosis of a bacterium into a host cell promotes cellular and genomic complexity. The mealybug Planococcus citri has two bacterial endosymbionts with an unusual nested arrangement: the γ-proteobacterium Moranella endobia lives in the cytoplasm of the ß-proteobacterium Tremblaya princeps These two bacteria, along with genes horizontally transferred from other bacteria to the P. citri genome, encode gene sets that form an interdependent metabolic patchwork. Here, we test the stability of this three-way symbiosis by sequencing host and symbiont genomes for five diverse mealybug species and find marked fluidity over evolutionary time. Although Tremblaya is the result of a single infection in the ancestor of mealybugs, the γ-proteobacterial symbionts result from multiple replacements of inferred different ages from related but distinct bacterial lineages. Our data show that symbiont replacement can happen even in the most intricate symbiotic arrangements and that preexisting horizontally transferred genes can remain stable on genomes in the face of extensive symbiont turnover.

Know your farmer: Ancient origins and multiple independent domestications of ambrosia beetle fungal cultivars.

Bark and ambrosia beetles are highly specialized weevils (Curculionidae) that have established diverse symbioses with fungi, most often from the order Ophiostomatales (Ascomycota, Sordariomycetes). The two types of beetles are distinguished by their feeding habits and intimacy of interactions with their symbiotic fungi. The tree tissue diet of bark beetles is facilitated by fungi, while ambrosia beetles feed solely on fungi that they farm. The farming life history strategy requires domestication of a fungus, which the beetles consume as their sole food source. Ambrosia beetles in the subfamily Platypodinae originated in the mid-Cretaceous (119-88 Ma) and are the oldest known group of farming insects. However, attempts to resolve phylogenetic relationships and the timing of domestication events for fungal cultivars have been largely inconclusive. We sequenced the genomes of 12 ambrosia beetle fungal cultivars and bark beetle associates, including the devastating laurel wilt pathogen, Raffaelea lauricola, to estimate a robust phylogeny of the Ophiostomatales. We find evidence for contemporaneous diversification of the beetles and their associated fungi, followed by three independent domestication events of the ambrosia fungi genus Raffaelea. We estimate the first domestication of an Ophiostomatales fungus occurred ~86 Ma, 25 million years earlier than prior estimates and in close agreement with the estimated age of farming in the Platypodinae (96 Ma). Comparisons of the timing of fungal domestication events with the timing of beetle radiations support the hypothesis that the first large beetle radiations may have spread domesticated "ambrosia" fungi to other fungi-associated beetle groups, perhaps facilitating the evolution of new farming lineages.

Genome expansion via lineage splitting and genome reduction in the cicada endosymbiont Hodgkinia.

Comparative genomics from mitochondria, plastids, and mutualistic endosymbiotic bacteria has shown that the stable establishment of a bacterium in a host cell results in genome reduction. Although many highly reduced genomes from endosymbiotic bacteria are stable in gene content and genome structure, organelle genomes are sometimes characterized by dramatic structural diversity. Previous results from Candidatus Hodgkinia cicadicola, an endosymbiont of cicadas, revealed that some lineages of this bacterium had split into two new cytologically distinct yet genetically interdependent species. It was hypothesized that the long life cycle of cicadas in part enabled this unusual lineage-splitting event. Here we test this hypothesis by investigating the structure of the Ca. Hodgkinia genome in one of the longest-lived cicadas, Magicicada tredecim. We show that the Ca. Hodgkinia genome from M. tredecim has fragmented into multiple new chromosomes or genomes, with at least some remaining partitioned into discrete cells. We also show that this lineage-splitting process has resulted in a complex of Ca. Hodgkinia genomes that are 1.1-Mb pairs in length when considered together, an almost 10-fold increase in size from the hypothetical single-genome ancestor. These results parallel some examples of genome fragmentation and expansion in organelles, although the mechanisms that give rise to these extreme genome instabilities are likely different.

Endosymbiosis: The feeling is not mutual.

Endosymbiosis is an idea that provided a remarkable amount of explanatory power about the origins of eukaryotic organelles. But it also promoted a number of assumptions that have also been influential, but are less well-examined. Here we look at two of these to see whether or not they fit current evidence. The assumption we first address is that endosymbiotic relationships such as nutritional symbioses and eukaryotic organelles are mutualisms. We argue instead that they are more one-sided associations that can be regarded as context-dependent power struggles like any other ecological interaction. The second assumption is that during endosymbiotic interactions (such as the origin of organelles), the host genomes will acquire a great many genes from endosymbionts that assume functions in host systems (as opposed to the well-documented genes whose products are simply targeted back to the endosymbiont or organelle). The idea that these genes exist in large numbers has been influential in a number of hypotheses about organelle evolution and distribution, but in the most carefully-examined systems no such mass migration of genes is evident. Overall, we argue that both the nature and impact of endosymbiosis need to be constantly re-evaluated to fully understand what roles it really plays in both cell biology and evolution.

Population genomics of a symbiont in the early stages of a pest invasion.

Invasive species often depend on microbial symbionts, but few studies have examined the evolutionary dynamics of symbionts during the early stages of an invasion. The insect Megacopta cribraria and its bacterial nutritional symbiont Candidatus Ishikawaella capsulata invaded the southeastern US in 2009. While M. cribraria was initially discovered on wild kudzu plants, it was found as a pest on soybeans within 1 year of infestation. Because prior research suggests Ishikawaella confers the pest status--that is, the ability to thrive on soybeans--in some Megacopta species, we performed a genomic study on Ishikawaella from US. Megacopta cribraria populations to understand the role of the symbiont in driving host plant preferences. We included Ishikawaella samples collected in the first days of the invasion in 2009 and from 23 locations across the insect's 2011 US range. The 0.75 Mb symbiont genome revealed only 47 fixed differences from the pest-conferring Ishikawaella in Japan, with only one amino acid change in a nutrition-provisioning gene. This similarity, along with a lack of fixed substitutions in the US symbiont population, indicates that Ishikawella likely arrived in the US capable of being a soybean pest. Analyses of allele frequency changes between 2009 and 2011 uncover signatures of both positive and negative selection and suggest that symbionts on soybeans and kudzu experience differential selection for genes related to nutrient provisioning. Our data reveal the evolutionary trajectory of an important insect-bacteria symbiosis in the early stages of an invasion, highlighting the role microbial symbionts may play in the spread of invasive species.

Dynamic recruitment of amino acid transporters to the insect/symbiont interface.

Symbiosis is well known to influence bacterial symbiont genome evolution and has recently been shown to shape eukaryotic host genomes. Intriguing patterns of host genome evolution, including remarkable numbers of gene duplications, have been observed in the pea aphid, a sap-feeding insect that relies on a bacterial endosymbiont for amino acid provisioning. Previously, we proposed that gene duplication has been important for the evolution of symbiosis based on aphid-specific gene duplication in amino acid transporters (AATs), with some paralogs highly expressed in the cells housing symbionts (bacteriocytes). Here, we use a comparative approach to test the role of gene duplication in enabling recruitment of AATs to bacteriocytes. Using genomic and transcriptomic data, we annotate AATs from sap-feeding and non sap-feeding insects and find that, like aphids, AAT gene families have undergone independent large-scale gene duplications in three of four additional sap-feeding insects. RNA-seq differential expression data indicate that, like aphids, the sap-feeding citrus mealybug possesses several lineage-specific bacteriocyte-enriched paralogs. Further, differential expression data combined with quantitative PCR support independent evolution of bacteriocyte enrichment in sap-feeding insect AATs. Although these data indicate that gene duplication is not necessary to initiate host/symbiont amino acid exchange, they support a role for gene duplication in enabling AATs to mediate novel host/symbiont interactions broadly in the sap-feeding suborder Sternorrhyncha. In combination with recent studies on other symbiotic systems, gene duplication is emerging as a general pattern in host genome evolution.

Retention of an Endosymbiont for the Production of a Single Molecule.

Sap-feeding insects often maintain two or more nutritional endosymbionts that act in concert to produce compounds essential for insect survival. Many mealybugs have endosymbionts in a nested configuration: one or two bacterial species reside within the cytoplasm of another bacterium, and together, these bacteria have genomes that encode interdependent sets of genes needed to produce key nutritional molecules. Here, we show that the mealybug Pseudococcus viburni has three endosymbionts, one of which contributes only two unique genes that produce the host nutrition-related molecule chorismate. All three bacterial endosymbionts have tiny genomes, suggesting that they have been coevolving inside their insect host for millions of years.