Genome-Wide Functional Divergence after the Symbiosis of Proteobacteria with Insects Unraveled through a Novel Computational Approach

Symbiosis has been among the most important evolutionary steps to generate biological complexity. The establishment of symbiosis required an intimate metabolic link between biological systems with different complexity levels. The strict endo-cellular symbiotic bacteria of insects are beautiful examples of the metabolic coupling between organisms belonging to different kingdoms, a eukaryote and a prokaryote. The host (eukaryote) provides the endosymbiont (prokaryote) with a stable cellular environment while the endosymbiont supplements the host's diet with essential metabolites. For such communication to take place, endosymbionts' genomes have suffered dramatic modifications and reconfigurations of proteins' functions. Two of the main modifications, loss of genes redundant for endosymbiotic bacteria or the host and bacterial genome streamlining, have been extensively studied. However, no studies have accounted for possible functional shifts in the endosymbiotic proteomes. Here, we develop a simple method to screen genomes for evidence of functional divergence between two species clusters, and we apply it to identify functional shifts in the endosymbiotic proteomes. Despite the strong effects of genetic drift in the endosymbiotic systems, we unexpectedly identified genes to be under stronger selective constraints in endosymbionts of aphids and ants than in their free-living bacterial relatives. These genes are directly involved in supplementing the host's diet with essential metabolites. A test of functional divergence supports a strong relationship between the endosymbiosis and the functional shifts of proteins involved in the metabolic communication with the insect host. The correlation between functional divergence in the endosymbiotic bacterium and the ecological requirements of the host uncovers their intimate biochemical and metabolic communication and provides insights on the role of symbiosis in generating species diversity

Introduction: symbiosis as a living evolving critique

Different species, interacting in a symbiotic fashion, living together over a prolonged period of time, eventually co-evolving into new species: this vision of the biological phenomenon of symbiosis has created a strong impression—both of symbiosis as a metaphor and a material reality—of species in an intimate relationship together, cooperating in spite of differences, of becoming something else and transgressing boundaries. This idea has turned the concept of symbiosis, in its many guises and definitions, into a breeding ground for a posthuman, biologically and ecologically informed critique. Less focused on the biological process of symbiosis as such, our focus in Symbiosis: Ecologies, Assemblages and Evolution is more on how symbiosis can be used as a means to argue for an alternative worldview and even a better world...

Purifying Selection and Molecular Adaptation in the Genome of Verminephrobacter, the Heritable Symbiotic Bacteria of Earthworms

While genomic erosion is common among intracellular symbionts, patterns of genome evolution in heritable extracellular endosymbionts remain elusive. We study vertically transmitted extracellular endosymbionts (Verminephrobacter, Betaproteobacteria) that form a beneficial, species-specific, and evolutionarily old (60–130 Myr) association with earthworms. We assembled a draft genome of Verminephrobacter aporrectodeae and compared it with the genomes of Verminephrobacter eiseniae and two nonsymbiotic close relatives (Acidovorax). Similar to V. eiseniae, the V. aporrectodeae genome was not markedly reduced in size and showed no A–T bias. We characterized the strength of purifying selection (ω = dN/dS) and codon usage bias in 876 orthologous genes. Symbiont genomes exhibited strong purifying selection (ω = 0.09 ± 0.07), although transition to symbiosis entailed relaxation of purifying selection as evidenced by 50% higher ω values and less codon usage bias in symbiont compared with reference genomes. Relaxation was not evenly distributed among functional gene categories but was overrepresented in genes involved in signal transduction and cell envelope biogenesis. The same gene categories also harbored instances of positive selection in the Verminephrobacter clade. In total, positive selection was detected in 89 genes, including also genes involved in DNA metabolism, tRNA modification, and TonB-dependent iron uptake, potentially highlighting functions important in symbiosis. Our results suggest that the transition to symbiosis was accompanied by molecular adaptation, while purifying selection was only moderately relaxed, despite the evolutionary age and stability of the host association. We hypothesize that biparental transmission of symbionts and rare genetic mixing during transmission can prevent genome erosion in heritable symbionts

Proteome-Wide Analysis of Functional Divergence in Bacteria: Exploring a Host of Ecological Adaptations

Functional divergence is the process by which new genes and functions originate through the modification of existing ones. Both genetic and environmental factors influence the evolution of new functions, including gene duplication or changes in the ecological requirements of an organism. Novel functions emerge at the expense of ancestral ones and are generally accompanied by changes in the selective forces at constrained protein regions. We present software capable of analyzing whole proteomes, identifying putative amino acid replacements leading to functional change in each protein and performing statistical tests on all tabulated data. We apply this method to 750 complete bacterial proteomes to identify high-level patterns of functional divergence and link these patterns to ecological adaptations. Proteome-wide analyses of functional divergence in bacteria with different ecologies reveal a separation between proteins involved in information processing (Ribosome biogenesis etc.) and those which are dependent on the environment (energy metabolism, defense etc.). We show that the evolution of pathogenic and symbiotic bacteria is constrained by their association with the host, and also identify unusual events of functional divergence even in well-studied bacteria such as Escherichia coli. We present a description of the roles of phylogeny and ecology in functional divergence at the level of entire proteomes in bacteria.This study was supported by a grant from the Spanish Ministerio de Ciencia e Inovacion (BFU2009-12022) and a grant of the Research Frontiers Program (10/RFP/GEN2685) from Science Foundation Ireland. 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Evolutionary Stability Of Fungal-Bacterial Endosymbioses

Many eukaryotes interact with heritable endobacteria to satisfy diverse metabolic needs. Of the characterized fungal-bacterial symbioses, endobacterial associations with the Gigasporaceae (Glomeromycota) and Rhizopus microsporus (Mucoromycotina) are the best described. Both fungal hosts associate with closely related bacterial endosymbionts from the Burkholderia lineage of [beta]-proteobacteria. Through investigating patterns of co-divergence between partners, we have shown that the Glomeribacter-Glomeromycota symbiosis is at least 400 million years old, while still remaining non-essential for the host. To further explore what adaptations have taken place to allow for the persistence of this association, we created a computational pipeline which utilizes patterns of adaptation to infer microbial lifestyle. We show that this pipeline is effective at inferring microbial lifestyle, and that genes involved in DNA regulation, energy metabolism, and pathogenicity are likely important for survival of Ca. Glomeribacter within their fungal hosts. Additionally, we identified that non-essential endosymbionts are as effective at purging slightly deleterious mutations from their genomes as free-living organisms. Unlike Glomeribacter, Burkholderia rhizoxinica, the endosymbiont of Rhizopus microsporus is capable of free living yet is simultaneously of great importance to host survival. Our work has revealed that endosymbionts are required for sexual reproduction of the fungal host. Through phenotypic observation and transcriptome profiling, we found that endosymbionts control fungal reproduction through hijacking of host reproductive machinery. Specifically, bacteria control expression levels of Ras2, a signaling protein important for reproductive development as well as filamentous growth. We also exploited endosymbiont control over reproduction to explore conservation of sexually relevant genes across Fungi, including the Mucoromycotina. This approach identified several genes that appear core to all fungal reproduction, as well as reproduction related genes which are specific to members of the Mucoromycotina. In particular, we found two candidate class C seven transmembrane G-protein coupled receptors (GPCRs), TriR1 and TriR2, which may be responsible for perception of trisporic acid during mating in Mucoromycotina. These receptors are closely related to the retinoic acid GPCRs present in animal systems

Ammonia-Oxidizing Archaea in Marine Cold-Water Sponges

Thaumarchaea (Archaea) kommen in vielen terrestrischen und marinen Lebensräumen vor und sind oftmals mit marinen Schwämmen assoziiert. Basierend auf Metagenom- und Kultivierungsstudien hat sich gezeigt, dass viele (wenn nicht sogar alle) Thaumarchaea die Fähigkeit besitzen, mittels dem Enzym Ammoniummonooxygenase (AMO), Ammonium zu oxidieren. In dieser Studie untersuchen wir die Aktivität und Diversität der Thaumarchaea in Kaltwasserschwämmen aus Norwegen. Durch die Kombination von quantitativ-physiologischen und molekularen Analysen und die Messungen von Produktion und Verbrauch bestimmter Stickstoffverbindungen in Inkubationsexperimenten wurde Nitrifikation in Geodia barretti, Phakellia ventilabrum, Antho dichotoma und Tentorium semisuberites nachgewiesen und quantifiziert. Zugleich konnte mittels quantitativer PCR und fluoreszenter in situ Hybridisierung eine hohe Anzahl an AMO-kodierenden Archaea (bis zu 6*108 archaeale amoA Genkopien pro µg Nukleinsäure) detektiert werden. Im Schwamm G. barretti wurden zum ersten Mal Raten von Denitrifikation und anaerober Ammoniumoxidation nachgewiesen und somit wurde gezeigt, dass der gesamten Stickstoffkreislauf in marinen Schwämmen ablaufen kann. Um die Diversität und Funktion der aktiven Archaea (und Bakterien) in G. barretti genauer zu untersuchen, haben wir den “Doppel-RNS” Metatranskriptomik Ansatz angewandt, wobei revers-transkribierte RNS direkt sequenziert und anschließend bioinformatisch ausgewertet wurde. Von den ca. 260.000 RNS-tags der Pyrosequenzierung, waren etwa 110.000 von der kleinen Untereinheit der ribosomalen RNS, woraus ein detailliertes taxonomisches Profil von den drei Domänen des Lebens des Schwammsystems erstellt wurde. Innerhalb der transkribierten mRNS-tags haben wir eine große Anzahl an archaealen Genen identifiziert, die wahrscheinlich in den Transport und die Oxidation von Ammonium involviert sind. Einige dieser hoch transkribierten Gene sind in thaumarchaealen Genomen konserviert, aber deren eventueller Beitrag in Ammoniumoxidation war bisher noch nicht bekannt. Aus den Messungen von Nitrifikationsraten, zusammen mit hoher Transkription von amoA Genen in mehreren Schwammarten aus der mesopelagischen Zone des Nordatlantiks, schließen wir eine Schlüsselrolle der Archaea für den Stickstoffmetabolismus mariner Schwämme.Thaumarchaeota have been discovered not only in a diverse range of moderate terrestrial and marine habitats but also as frequent inhabitants of marine sponges. Based on metagenomic and cultivation studies, it has become evident over the past years that some (or all) thaumarchaea have the capability of oxidizing ammonia using the enzyme ammonia monooxygenase (Amo), a homologue of the well-known bacterial counterpart. Here we explore the activity and diversity of Thaumarchaeota in marine cold-water sponges of the Northern hemisphere (Norway) by combining quantitative physiological data and molecular analyses. By monitoring the production and consumption of nitrogen compounds in defined incubation experiments we have demonstrated and quantified nitrification in Geodia barretti, Phakellia ventilabrum, Tentorium semisuberites and Antho dichotoma. In parallel, large numbers of Amo-encoding archaea were detected by quantitative PCR (up to 6*108 archaeal amoA gene copies per µg of nucleic acids) and fluorescence in situ hybridisation, with bacterial amoA genes mostly being under the detection limit. We report denitrification and anammox rates in the sponge Geodia barretti beside nitrification activity by employing stable isotope labelling techniques, thus closing the nitrogen cycle in a marine sponge for the first time. We also identified the potential microbial lineages that are responsible for the activities. To obtain insights into the in situ diversity and function of active microbes in Geodia barretti we employed the “double-RNA” approach, which involved analysis of reverse-transcribed total RNA. Of the approximately 260,000 RNA-tags obtained by pyrosequencing, we assigned ≈110,000 tags to small subunit rRNA and derived a detailed community profile of all three domains of life. Around 50% of all 16S rRNA-tags were assembled and phylogeny of the abundant taxa was performed and compared to sequences of a 16S rRNA clone library of the same cDNA. Within the expressed sequence tags (mRNA), we identified a large number of archaeal genes that are potentially involved in transport and oxidation of ammonia. Some of these highly expressed genes are conserved in thaumarchaeal genomes but their potential function in ammonia oxidation was not previously recognized. From our studies we infer a key role for archaea in the nitrogen metabolism in marine sponges

Molecular Evolution of Metazoan Hypoxia-inducing Factors

Most metazoans rely on aerobic energy production, which is dependent on adequate oxygen supply. In the case of reduced oxygen supply (hypoxia), the most profound changes in gene expression are mediated by transcription factors named hypoxia-inducible factors (HIF alpha). These proteins are post-translationally regulated by prolyl-4-hydroxylase (PHD) enzymes that are direct “sensors” of cellular oxygen levels. This thesis examines the molecular evolution of metazoan HIF systems. In early metazoans the HIF system emerged from pre-existing PHD oxygen sensors and early bHLH-PAS transcription factors. In invertebrates our analysis revealed an unexpected diversity of PHD genes and HIF alpha sequence characteristics. An early branching vertebrate, the epaulette shark (Hemiscyllium ocellatum) was chosen for sequencing and hypoxia preconditioning studies of HIF alpha and PHD genes. As no quantitative PCR reference genes were available, this thesis includes the first study of reference genes in cartilaginous fish species. Applying multiple statistical analysis we also discoveredthat commonly used reference gene software may perform poorly with some data sets. Novel reference genes allowed accurate measurements of the mRNAlevels of the studied target genes. Cartilaginous fishes have three genomic duplicates of both HIF alpha and PHD genes like mammals and teleost fishes. Combining functional divergence and selection analyses it was possible to describe how sequence changes in both HIF alpha and PHD duplicates may have contributed to the differential oxygen sensitivityof HIF alphas. Additionally, novel teleost HIF-1 alpha sequences were produced and used to reveal the molecular evolution of HIF-1 alpha in this lineage rich with hypoxia tolerant species.Siirretty Doriast

Reticulate Evolution: Symbiogenesis, Lateral Gene Transfer, Hybridization and Infectious heredity

info:eu-repo/semantics/publishedVersio

Comparative Genomics Between Saccharomyces kudriavzevii and S. cerevisiae Applied to Identify Mechanisms Involved in Adaptation

Yeasts belonging to the Saccharomyces genus play an important role in human-driven fermentations. The species S. cerevisiae has been widely studied because it is the dominant yeast in most fermentations and it has been widely used as a model eukaryotic organism. Recently, other species of the Saccharomyces genus are gaining interest to solve the new challenges that the fermentation industry are facing. One of these species is S. kudriavzevii, which exhibits interesting physiological properties compared to S. cerevisiae, such as a better adaptation to grow at low temperatures, a higher glycerol synthesis and lower ethanol production. The aim of this study is to understand the molecular basis behind these phenotypic differences of biotechnological interest by using a species-based comparative genomics approach. In this work, we sequenced, assembled and annotated two new genomes of S. kudriavzevii. We used a combination of different statistical methods to identify functional divergence, signatures of positive selection and acceleration of substitution rates at specific amino acid sites of proteins in S. kudriavzevii when compared to S. cerevisiae, and vice versa. We provide a list of candidate genes in which positive selection could be acting during the evolution of both S. cerevisiae and S. kudriavzevii clades. Some of them could be related to certain important differences in metabolism previously reported by other authors such us DAL3 and ARO4, involved in nitrogen assimilation and amino acid biosynthesis. In addition, three of those genes (FBA1, ZIP1, and RQC2) showed accelerated evolutionary rates in Sk branch. Finally, genes of the riboflavin biosynthesis were also among those genes with a significant higher rate of nucleotide substitution and those proteins have amino acid positions contributing to functional divergence

Infections, Symbiosis, Immunity and Adaptation

Evolution has been shaping the genetic structure of populations across generations, using mutation and recombination, migration, drift and selection to create and/or corrode variation. The array of traits presented by individuals in a population is dependent on several factors, such as their heritability or the genetic pool available to the adaptive process. Additionally, the multitude of complex relationships within and between species creates another level of complexity that can compromise the pinpointing of the contributing factors and their relative weight to such changes. As so, understandably, disentangling the factors that influence the course of evolution in natural populations is of extreme importance but also of great difficulty. (...