Stochasticity, determinism, and contingency shape genome evolution of endosymbiotic bacteria.

Evolution results from the interaction of stochastic and deterministic processes that create a web of historical contingency, shaping gene content and organismal function. To understand the scope of this interaction, we examine the relative contributions of stochasticity, determinism, and contingency in shaping gene inactivation in 34 lineages of endosymbiotic bacteria, Sodalis, found in parasitic lice, Columbicola, that are independently undergoing genome degeneration. Here we show that the process of genome degeneration in this system is largely deterministic: genes involved in amino acid biosynthesis are lost while those involved in providing B-vitamins to the host are retained. In contrast, many genes encoding redundant functions, including components of the respiratory chain and DNA repair pathways, are subject to stochastic loss, yielding historical contingencies that constrain subsequent losses. Thus, while selection results in functional convergence between symbiont lineages, stochastic mutations initiate distinct evolutionary trajectories, generating diverse gene inventories that lack the functional redundancy typically found in free-living relatives.

Protocol to establish a genetically tractable synthetic symbiosis between Sodalis praecaptivus and grain weevils by insect egg microinjection.

We present a protocol to establish a synthetic symbiosis between the mCherry-expressing Sodalis praecaptivus and the grain weevil host, Sitophilus zeamais. We describe steps to isolate grain weevil eggs, followed by microinjecting the bacterial symbiont into insect eggs using a modified Drosophila injection protocol, which leads to localization of bacteria in female insect ovaries. We then detail larval transplantation and visualization of bacteria in live insects using a fluorescence dissection microscope to assess the transgenerational transmission to offspring in weevils. For complete details on the use and execution of this protocol, please refer to Su et al. (2022).1.

Rational engineering of a synthetic insect-bacterial mutualism.

Many insects maintain mutualistic associations with bacterial endosymbionts, but little is known about how they originate in nature. In this study, we describe the establishment and manipulation of a synthetic insect-bacterial symbiosis in a weevil host. Following egg injection, the nascent symbiont colonized many tissues, including prototypical somatic and germinal bacteriomes, yielding maternal transmission over many generations. We then engineered the nascent symbiont to overproduce the aromatic amino acids tyrosine and phenylalanine, which facilitate weevil cuticle strengthening and accelerated larval development, replicating the function of mutualistic symbionts that are widely distributed among weevils and other beetles in nature. Our work provides empirical support for the notion that mutualistic symbioses can be initiated in insects by the acquisition of environmental bacteria. It also shows that certain bacterial genera, including the Sodalis spp. used in our study, are predisposed to develop these associations due to their ability to maintain benign infections and undergo vertical transmission in diverse insect hosts, facilitating the partner-fidelity feedback that is critical for the evolution of obligate mutualism. These experimental advances provide a new platform for laboratory studies focusing on the molecular mechanisms and evolutionary processes underlying insect-bacterial symbiosis.

Microbiome stability and structure is governed by host phylogeny over diet and geography in woodrats (Neotoma spp.).

The microbiome is critical for host survival and fitness, but gaps remain in our understanding of how this symbiotic community is structured. Despite evidence that related hosts often harbor similar bacterial communities, it is unclear whether this pattern is due to genetic similarities between hosts or to common ecological selection pressures. Here, using herbivorous rodents in the genus Neotoma, we quantify how geography, diet, and host genetics, alongside neutral processes, influence microbiome structure and stability under natural and captive conditions. Using bacterial and plant metabarcoding, we first characterized dietary and microbiome compositions for animals from 25 populations, representing seven species from 19 sites across the southwestern United States. We then brought wild animals into captivity, reducing the influence of environmental variation. In nature, geography, diet, and phylogeny collectively explained ∼50% of observed microbiome variation. Diet and microbiome diversity were correlated, with different toxin-enriched diets selecting for distinct microbial symbionts. Although diet and geography influenced natural microbiome structure, the effects of host phylogeny were stronger for both wild and captive animals. In captivity, gut microbiomes were altered; however, responses were species specific, indicating again that host genetic background is the most significant predictor of microbiome composition and stability. In captivity, diet effects declined and the effects of host genetic similarity increased. By bridging a critical divide between studies in wild and captive animals, this work underscores the extent to which genetics shape microbiome structure and stability in closely related hosts.

Incipient genome erosion and metabolic streamlining for antibiotic production in a defensive symbiont.

Genome erosion is a frequently observed result of relaxed selection in insect nutritional symbionts, but it has rarely been studied in defensive mutualisms. Solitary beewolf wasps harbor an actinobacterial symbiont of the genus Streptomyces that provides protection to the developing offspring against pathogenic microorganisms. Here, we characterized the genomic architecture and functional gene content of this culturable symbiont using genomics, transcriptomics, and proteomics in combination with in vitro assays. Despite retaining a large linear chromosome (7.3 Mb), the wasp symbiont accumulated frameshift mutations in more than a third of its protein-coding genes, indicative of incipient genome erosion. Although many of the frameshifted genes were still expressed, the encoded proteins were not detected, indicating post-transcriptional regulation. Most pseudogenization events affected accessory genes, regulators, and transporters, but "Streptomyces philanthi" also experienced mutations in central metabolic pathways, resulting in auxotrophies for biotin, proline, and arginine that were confirmed experimentally in axenic culture. In contrast to the strong A+T bias in the genomes of most obligate symbionts, we observed a significant G+C enrichment in regions likely experiencing reduced selection. Differential expression analyses revealed that-compared to in vitro symbiont cultures-"S. philanthi" in beewolf antennae showed overexpression of genes for antibiotic biosynthesis, the uptake of host-provided nutrients and the metabolism of building blocks required for antibiotic production. Our results show unusual traits in the early stage of genome erosion in a defensive symbiont and suggest tight integration of host-symbiont metabolic pathways that effectively grants the host control over the antimicrobial activity of its bacterial partner.

Quorum sensing sets the stage for the establishment and vertical transmission of Sodalis praecaptivus in tsetse flies.

Bacterial virulence factors facilitate host colonization and set the stage for the evolution of parasitic and mutualistic interactions. The Sodalis-allied clade of bacteria exhibit striking diversity in the range of both plant and animal feeding insects they inhabit, suggesting the appropriation of universal molecular mechanisms that facilitate establishment. Here, we report on the infection of the tsetse fly by free-living Sodalis praecaptivus, a close relative of many Sodalis-allied symbionts. Key genes involved in quorum sensing, including the homoserine lactone synthase (ypeI) and response regulators (yenR and ypeR) are integral for the benign colonization of S. praecaptivus. Mutants lacking ypeI, yenR and ypeR compromised tsetse survival as a consequence of their inability to repress virulence. Genes under quorum sensing, including homologs of the binary insecticidal toxin PirAB and a putative symbiosis-promoting factor CpmAJ, demonstrated negative and positive impacts, respectively, on tsetse survival. Taken together with results obtained from experiments involving weevils, this work shows that quorum sensing virulence suppression plays an integral role in facilitating the establishment of Sodalis-allied symbionts in diverse insect hosts. This knowledge contributes to the understanding of the early evolutionary steps involved in the formation of insect-bacterial symbiosis. Further, despite having no established history of interaction with tsetse, S. praecaptivus can infect reproductive tissues, enabling vertical transmission through adenotrophic viviparity within a single host generation. This creates an option for the use of S. praecaptivus in the biocontrol of insect disease vectors via paratransgenesis.

The Life of an Insect Endosymbiont from the Cradle to the Grave.

Host-beneficial endosymbioses, which are formed when a microorganism takes up residence inside another cell and provides a fitness advantage to the host, have had a dramatic influence on the evolution of life. These intimate relationships have yielded the mitochondrion and the plastid (chloroplast) - the ancient organelles that in part define eukaryotic life - along with many more recent associations involving a wide variety of hosts and microbial partners. These relationships are often envisioned as stable associations that appear cooperative and persist for extremely long periods of time. But recent evidence suggests that this stable state is often born from turbulent and conflicting origins, and that the apparent stability of many beneficial endosymbiotic relationships - although certainly real in many cases - is not an inevitable outcome of these associations. Here we review how stable endosymbioses form, how they are maintained, and how they sometimes break down and are reborn. We focus on relationships formed by insects and their resident microorganisms because these symbioses have been the focus of significant empirical work over the last two decades. We review these relationships over five life stages: origin, birth, middle age, old age, and death.

Metagenomic sequencing provides insights into microbial detoxification in the guts of small mammalian herbivores (Neotoma spp.).

Microbial detoxification of plant toxins influences the use of plants as food sources by herbivores. Stephen's woodrats (Neotoma stephensi) specialize on juniper, which is defended by oxalate, phenolics and monoterpenes, while closely related N. albigula specialize on cactus, which only contains oxalate. Woodrats maintain two gut chambers harboring dense microbial communities: a foregut chamber proximal to the major site of toxin absorption, and a cecal chamber in their hindgut. We performed several experiments to investigate the location and nature of microbial detoxification in the woodrat gut. First, we measured toxin concentrations across gut chambers of N. stephensi. Compared to food material, oxalate concentrations were immediately lower in the foregut, while concentrations of terpenes remained high in the foregut, and were lowest in the cecal chamber. We conducted metagenomic sequencing of the foregut chambers of both woodrat species and cecal chambers of N. stephensi to compare microbial functions. We found that most genes associated with detoxification were more abundant in the cecal chambers of N. stephensi. However, some genes associated with degradation of oxalate and phenolic compounds were more abundant in the foregut chambers. Thus, microbial detoxification may take place in various chambers depending on the class of chemical compound.

Evolution: Weevils Get Tough on Symbiotic Tyrosine.

Weevils, which represent one of the most diverse groups of terrestrial insects in nature, obtain a tough exoskeleton through the activity of an ancient bacterial symbiont with a tiny genome that serves as a factory for the production of tyrosine.

Microbiota Diversification and Crash Induced by Dietary Oxalate in the Mammalian Herbivore Neotoma albigula.

Oxalate, broadly found in both dietary and endogenous sources, is a primary constituent in 80% of kidney stones, an affliction that has tripled in prevalence over the last 40 years. Oxalate-degrading bacteria within the gut microbiota can mitigate the effects of oxalate and are negatively correlated with kidney stone formation, but bacteriotherapies involving oxalate-degrading bacteria have met with mixed results. To inform the development of more effective and consistent bacteriotherapies, we sought to quantify the interactions and limits between oxalate and an oxalate-adapted microbiota from the wild mammalian herbivore Neotoma albigula (woodrat), which consumes a high-oxalate diet in the wild. We tracked the microbiota over a variable-oxalate diet ranging from 0.2% to 12%, with the upper limit approximating 10× the level of human consumption. The N. albigula microbiota was capable of degrading ~100% of dietary oxalate regardless of the amount consumed. However, the microbiota exhibited significant changes in diversity dynamically at the operational taxonomic unit (OTU), family, and community levels in accordance with oxalate input. Furthermore, a cohesive microbial network was stimulated by the consumption of oxalate and exhibited some resistance to the effects of prolonged exposure. This study demonstrates that the oxalate-adapted microbiota of N. albigula exhibits a very high level of degradation and tolerance for oxalate. IMPORTANCE The bacteria associated with mammalian hosts exhibit extensive interactions with overall host physiology and contribute significantly to the health of the host. Bacteria are vital to the mitigation of the toxic effects of oxalate specifically as mammals do not possess the enzymes to degrade this compound, which is present in the majority of kidney stones. Contrary to the body of literature on a few oxalate-degrading specialists, our work illustrates that oxalate stimulates a broad but cohesive microbial network in a dose-dependent manner. The unique characteristics of the N. albigula microbiota make it an excellent source for the development of bacteriotherapies to inhibit kidney stone formation. Furthermore, this work successfully demonstrates methods to identify microbial networks responsive to specific toxins, their limits, and important elements such as microbial network cohesivity and architecture. These are necessary steps in the development of targeted bacteriotherapies.

Patterns of host gene expression associated with harboring a foregut microbial community.

BACKGROUND: Harboring foregut microbial communities is considered a key innovation that allows herbivorous mammals to colonize new ecological niches. However, the functions of these chambers have only been well studied at the molecular level in ruminants. Here, we investigate gene expression in the foregut chamber of herbivorous rodents and ask whether these gene expression patterns are consistent with results in ruminants. We compared gene expression in foregut tissues of two rodent species: Stephen's woodrat (Neotoma stephensi), which harbors a dense foregut microbial community, and the lab rat (Rattus norvegicus), which lacks such a community. RESULTS: We found that woodrats have higher abundances of transcripts associated with smooth muscle processes, specifically a higher expression of the smoothelin-like 1 gene, which may assist in contractile properties of this tissue to retain food material in the foregut chamber. The expression of genes associated with keratinization and cornification exhibited a complex pattern of differences between the two species, suggesting distinct molecular mechanisms. Lab rats exhibited higher abundances of transcripts associated with immune function, likely to inhibit microbial growth in the foregut of this species. CONCLUSIONS: Some of our results were consistent with previous findings in ruminants (high expression of facilitative glucose transporters, lower expression of B4galnt2), suggestive of possible convergent evolution, while other results were unclear, and perhaps represent novel host-microbe interactions in rodents. Overall, our results suggest that harboring a foregut microbiota is associated with changes to the functions and host-microbe interactions of the foregut tissues.

The Induction of Oxalate Metabolism In Vivo Is More Effective with Functional Microbial Communities than with Functional Microbial Species.

For mammals, oxalate enters the body through the diet or is endogenously produced by the liver; it is removed by microbial oxalate metabolism in the gut and/or excretion in feces or urine. Deficiencies in any one of the these pathways can lead to complications, such as calcium oxalate urinary stones. While considerable research has been conducted on individual oxalate-degrading bacterial isolates, interactions between oxalate and the gut microbiota as a whole are unknown. We examined the reduction in oxalate excretion in a rat model following oral administration of fecal microbes from a mammalian herbivore adapted to a high oxalate diet or to fecal transplants consisting of two different formulations of mixed oxalate-degrading isolates. While all transplants elicited a significant reduction in oxalate excretion initially, the greatest effect was seen with fecal microbial transplants, which persisted even in the absence of dietary oxalate. The reduction in oxalate excretion in animals given fecal transplants corresponded with the establishment of diverse bacteria, including known oxalate-degrading bacteria and a cohesive network of bacteria centered on oxalate-degrading specialists from the Oxalobacteraceae family. Results suggested that the administration of a complete community of bacteria facilitates a cohesive balance in terms of microbial interactions. Our work offers important insights into the development of targeted bacteriotherapies intended to reduce urinary oxalate excretion in patients at risk for recurrent calcium oxalate stones as well as bacteriotherapies targeting other toxins for elimination. IMPORTANCE Oxalate is a central component in 80% of kidney stones. While mammals do not possess the enzymes to degrade oxalate, many gastrointestinal bacteria are efficient oxalate degraders. We examined the role of cohesive microbial networks for oxalate metabolism, using Sprague-Dawley rats as a model host. While the transplantation of oxalate-degrading bacteria alone to the Sprague-Dawley hosts did increase oxalate metabolism, fecal transplants from a wild mammalian herbivore, Neotoma albigula, had a significantly greater effect. Furthermore, the boost for oxalate metabolism persisted only in animals that received fecal transplants. Animals receiving fecal transplants had a more diverse and cohesive network of bacteria associated with the Oxalobacteraceae, a family known to consist of specialist oxalate-degrading bacteria, than did animals that received oxalate-degrading bacteria alone. Our results indicate that fecal transplants are more effective at transferring specific functions than are microbial specialists alone, which has broad implications for the development of bacteriotherapies.

Quorum Sensing Attenuates Virulence in Sodalis praecaptivus.

Sodalis praecaptivus is a close relative and putative environmental progenitor of the widely distributed, insect-associated, Sodalis-allied symbionts. Here we show that mutant strains of S. praecaptivus that lack genetic components of a quorum-sensing (QS) apparatus have a rapid and potent killing phenotype following microinjection into an insect host. Transcriptomic and genetic analyses indicate that insect killing occurs as a consequence of virulence factors, including insecticidal toxins and enzymes that degrade the insect integument, which are normally repressed by QS at high infection densities. This method of regulation suggests that virulence factors are only utilized in early infection to initiate the insect-bacterial association. Once bacteria reach sufficient density in host tissues, the QS circuit represses expression of these harmful genes, facilitating a long-lasting and benign association. We discuss the implications of the functionality of this QS system in the context of establishment and evolution of mutualistic relationships involving these bacteria.

The regulation of antimicrobial peptide resistance in the transition to insect symbiosis.

Many bacteria utilize two-component systems consisting of a sensor kinase and a transcriptional response regulator to detect environmental signals and modulate gene expression for adaptation. The response regulator PhoP and its cognate sensor kinase PhoQ compose a two-component system known for its role in responding to low levels of Mg2+ , Ca2+ , pH and to the presence of antimicrobial peptides and activating the expression of genes involved in adaptation to host association. Compared with their free-living relatives, mutualistic insect symbiotic bacteria inhabit a static environment where the requirement for sensory functions is expected to be relaxed. The insect symbiont, Sodalis glossinidius, requires PhoP to resist killing by host derived antimicrobial peptides. However, the S. glossinidius PhoQ was found to be insensitive to Mg2+ , Ca2+ and pH. Here they show that Sodalis praecaptivus, a close non host-associated relative of S. glossinidius, utilizes a magnesium sensing PhoP-PhoQ and an uncharacterized MarR-like transcriptional regulator (Sant_4061) to control antimicrobial peptide resistance in vitro. While the inactivation of phoP, phoQ or Sant_4061 completely retards the growth of S. praecaptivus in the presence of an antimicrobial peptide in vitro, inactivation of both phoP and Sant_4061 is necessary to abrogate growth of this bacterium in an insect host.

Microbial Community Transplant Results in Increased and Long-Term Oxalate Degradation.

Gut microbes are essential for the degradation of dietary oxalate, and this function may play a role in decreasing the incidence of kidney stones. However, many oxalate-degrading bacteria are susceptible to antibiotics and the use of oxalate-degrading probiotics has only led to an ephemeral reduction in urinary oxalate. The objective of the current study was to determine the efficacy of using whole-community microbial transplants from a wild mammalian herbivore, Neotoma albigula, to increase oxalate degradation over the long term in the laboratory rat, Rattus norvegicus. We quantified the change in total oxalate degradation in lab rats immediately after microbial transplants and at 2- and 9-month intervals following microbial transplants. Additionally, we tracked the fecal microbiota of the lab rats, with and without microbial transplants, using high-throughput Illumina sequencing of a hyper-variable region of the 16S rRNA gene. Microbial transplants resulted in a significant increase in oxalate degradation, an effect that persisted 9 months after the initial transplants. Functional persistence was corroborated by the transfer, and persistence of a group of bacteria previously correlated with oxalate consumption in N. albigula, including an anaerobic bacterium from the genus Oxalobacter known for its ability to use oxalate as a sole carbon source. The results of this study indicate that whole-community microbial transplants are an effective means for the persistent colonization of oxalate-degrading bacteria in the mammalian gut.

Draft Genome Sequence of an Oxalate-Degrading Strain of Clostridium sporogenes from the Gastrointestinal Tract of the White-Throated Woodrat (Neotoma albigula).

The gastrointestinal tract of the white-throated woodrat Neotoma albigula harbors a diverse microbial population that functions in the degradation of ingested plant secondary compounds. Here, we present the draft genome sequence and annotation of Clostridium sporogenes strain 8-O, a novel oxalate-degrading bacterium isolated from the feces of N. albigula.

Adaptation by Deletogenic Replication Slippage in a Nascent Symbiont.

As a consequence of population level constraints in the obligate, host-associated lifestyle, intracellular symbiotic bacteria typically exhibit high rates of molecular sequence evolution and extensive genome degeneration over the course of their host association. While the rationale for genome degeneration is well understood, little is known about the molecular mechanisms driving this change. To understand these mechanisms we compared the genome of Sodalis praecaptivus, a nonhost associated bacterium that is closely related to members of the Sodalis-allied clade of insect endosymbionts, with the very recently derived insect symbiont Candidatus Sodalis pierantonius. The characterization of indel mutations in the genome of Ca Sodalis pierantonius shows that the replication system in this organism is highly prone to deletions resulting from polymerase slippage events in regions encoding G+C-rich repetitive sequences. This slippage-prone phenotype is mechanistically associated with the loss of certain components of the bacterial DNA recombination machinery at an early stage in symbiotic life and is expected to facilitate rapid adaptation to the novel host environment. This is analogous to the emergence of mutator strains in both natural and laboratory populations of bacteria, which tend to reach high frequencies in clonal populations due to linkage between the mutator allele and the resulting adaptive mutations.

Effect of Dietary Oxalate on the Gut Microbiota of the Mammalian Herbivore Neotoma albigula.

Diet is one of the primary drivers that sculpts the form and function of the mammalian gut microbiota. However, the enormous taxonomic and metabolic diversity held within the gut microbiota makes it difficult to isolate specific diet-microbe interactions. The objective of the current study was to elucidate interactions between the gut microbiota of the mammalian herbivore Neotoma albigula and dietary oxalate, a plant secondary compound (PSC) degraded exclusively by the gut microbiota. We quantified oxalate degradation in N. albigula fed increasing amounts of oxalate over time and tracked the response of the fecal microbiota using high-throughput sequencing. The amount of oxalate degraded in vivo was linearly correlated with the amount of oxalate consumed. The addition of dietary oxalate was found to impact microbial species diversity by increasing the representation of certain taxa, some of which are known to be capable of degrading oxalate (e.g., Oxalobacter spp.). Furthermore, the relative abundances of 117 operational taxonomic units (OTU) exhibited a significant correlation with oxalate consumption. The results of this study indicate that dietary oxalate induces complex interactions within the gut microbiota that include an increase in the relative abundance of a community of bacteria that may contribute either directly or indirectly to oxalate degradation in mammalian herbivores.

Phenotypic characterization of Sodalis praecaptivus sp. nov., a close non-insect-associated member of the Sodalis-allied lineage of insect endosymbionts.

A Gram-stain-negative bacterium, isolated from a human wound was previously found to share an unprecedentedly close relationship with Sodalis glossinidius and other members of the Sodalis-allied clade of insect symbionts. This relationship was inferred from sequence analysis of the 16S rRNA gene and genomic comparisons and suggested the strain belonged to a novel species. Biochemical and genetic analyses supported this suggestion and demonstrated that the organism has a wide repertoire of metabolic properties, which is consistent with the presence of a relatively large gene inventory. Among members of the Sodalis-allied clade, this is the first representative that has sufficient metabolic capabilities to sustain growth in minimal media. On the basis of the results of this study, we propose that this organism be classified as a representative of a novel species, Sodalis praecaptivus sp. nov. (type strain HS(T) = DSM 27494(T) = ATCC BAA-2554(T)).

Gut microbes of mammalian herbivores facilitate intake of plant toxins.

The foraging ecology of mammalian herbivores is strongly shaped by plant secondary compounds (PSCs) that defend plants against herbivory. Conventional wisdom holds that gut microbes facilitate the ingestion of toxic plants; however, this notion lacks empirical evidence. We investigated the gut microbiota of desert woodrats (Neotoma lepida), some populations of which specialise on highly toxic creosote bush (Larrea tridentata). Here, we demonstrate that gut microbes are crucial in allowing herbivores to consume toxic plants. Creosote toxins altered the population structure of the gut microbiome to facilitate an increase in abundance of genes that metabolise toxic compounds. In addition, woodrats were unable to consume creosote toxins after the microbiota was disrupted with antibiotics. Last, ingestion of toxins by naïve hosts was increased through microbial transplants from experienced donors. These results demonstrate that microbes can enhance the ability of hosts to consume PSCs and therefore expand the dietary niche breadth of mammalian herbivores.