Unique mobile elements and scalable gene flow at the prokaryote–eukaryote boundary revealed by circularized Asgard archaea genomes

Eukaryotic genomes are known to have garnered innovations from both archaeal and bacterial domains but the sequence of events that led to the complex gene repertoire of eukaryotes is largely unresolved. Here, through the enrichment of hydrothermal vent microorganisms, we recovered two circularized genomes of Heimdallarchaeum species that belong to an Asgard archaea clade phylogenetically closest to eukaryotes. These genomes reveal diverse mobile elements, including an integrative viral genome that bidirectionally replicates in a circular form and aloposons, transposons that encode the 5,000 amino acid-sized proteins Otus and Ephialtes. Heimdallaechaeal mobile elements have garnered various genes from bacteria and bacteriophages, likely playing a role in shuffling functions across domains. The number of archaea- and bacteria-related genes follow strikingly different scaling laws in Asgard archaea, exhibiting a genome size-dependent ratio and a functional division resembling the bacteria- and archaea-derived gene repertoire across eukaryotes. Bacterial gene import has thus likely been a continuous process unaltered by eukaryogenesis and scaled up through genome expansion. Our data further highlight the importance of viewing eukaryogenesis in a pan-Asgard context, which led to the proposal of a conceptual framework, that is, the Heimdall nucleation–decentralized innovation–hierarchical import model that accounts for the emergence of eukaryotic complexity.

Syntrophic propionate-oxidizing bacteria in methanogenic systems

The mutual nutritional cooperation underpinning syntrophic propionate degradation provides a scant amount of energy for the microorganisms involved, so propionate degradation often acts as a bottleneck in methanogenic systems. Understanding the ecology, physiology, and metabolic capacities of syntrophic propionate-oxidizing bacteria is of interest in both engineered and natural ecosystems, as it offers prospects to guide further development of technologies for biogas production and biomass-derived chemicals, and is important in forecasting contributions by biogenic methane emissions to climate change. Syntrophic propionate-oxidizing bacteria are distributed across different phyla. They can exhibit broad metabolic capabilities in addition to syntrophy (e.g. fermentative, sulfidogenic, and acetogenic metabolism) and demonstrate variations in interplay with cooperating partners, indicating nuances in their syntrophic lifestyle. In this review, we discuss distinctions in gene repertoire and organization for the methylmalonyl-CoA pathway, hydrogenases and formate dehydrogenases, and emerging facets of (formate/hydrogen/direct) electron transfer mechanisms. We also use information from cultivations, thermodynamic calculations, and omic analyses as the basis for identifying environmental conditions governing propionate oxidation in various ecosystems. Overall, this review improves basic and applied understanding of syntrophic propionate-oxidizing bacteria and highlights knowledge gaps, hopefully encouraging future research and engineering on propionate metabolism in biotechnological processes.

Evolution of the Parvalbumin Genes in Teleost Fishes after the Whole-Genome Duplication

Parvalbumin is considered a major fish allergen. Here, we report the molecular evolution of the parvalbumin genes in bony fishes based on 19 whole genomes and 70 transcriptomes. We found unexpectedly high parvalbumin diversity in teleosts; three main gene types (pvalb-α, pvalb-β1, and pvalb-β2, including oncomodulins) originated at the onset of vertebrates. Teleosts have further multiplied the parvalbumin gene repertoire up to nine ancestral copies—two copies of pvalb-α, two copies of pvalb-β1, and five copies of pvalb-β2. This gene diversity is a result of teleost-specific whole-genome duplication. Two conserved parvalbumin genomic clusters carry pvalb-β1 and β2 copies, whereas pvalb-α genes are located separately in different linkage groups. Further, we investigated parvalbumin gene expression in 17 tissues of the common carp (Cyprinus carpio), a species with 21 parvalbumin genes in its genome. Two pvalb-α and eight pvalb-β2 copies are highly expressed in the muscle, while two alternative pvalb-α copies show expression in the brain and the testes, and pvalb-β1 is dominant in the retina and the kidney. The recent pairs of muscular pvalb-β2 genes show differential expression in this species. We provide robust genomic evidence of the complex evolution of the parvalbumin genes in fishes.

Genome of the bi-annual Rio Pearlfish (Nematolebias whitei), a killifish model species for Eco-Evo-Devo

The Rio Pearlfish Nematolebias whitei is a bi-annual killifish species inhabiting seasonal pools of the Rio de Janeiro region that dry twice per year. Embryos enter dormant diapause stages in the soil, waiting for the inundation of the habitat which triggers hatching and commencement of a new life cycle. This species represents a convergent, independent origin of annualism from other emerging killifish model species. While some transcriptomic datasets are available for Rio Pearlfish, thus far a sequenced genome has been unavailable. Here we present a high quality, 1.2Gb chromosome-level genome assembly, genome annotations and a comparative genomic investigation of the Rio Pearlfish as representative of a vertebrate clade that evolved environmentally-cued hatching. We show conservation of 3-D genome structure across teleost fish evolution, developmental stages, tissues and cell types. Our analysis of mobile DNA shows that Rio Pearlfish, like other annual killifishes, possesses an expanded transposable element profile with implications for rapid aging and adaptation to harsh conditions. We use the Rio Pearlfish genome to identify its hatching enzyme gene repertoire and the location of the hatching gland, a key first step in understanding the developmental genetic control of hatching. The Rio Pearlfish genome expands the comparative genomic toolkit available to study convergent origins of seasonal life histories, diapause, and rapid aging phenotypes. We present the first set of genomic resources for this emerging model organism, critical for future functional genetic and multi-omic explorations of Eco-Evo-Devo phenotypes in resilience and adaptation to extreme environments.

Comparative Genomics and Pathogenicity Analysis of Two Bacterial Symbionts of Entomopathogenic Nematodes: The Role of The GroEL Protein in Virulence

Bacteria of the genera Xenorhabdus and Photorhabdus are symbionts of entomopathogenic nematodes. Despite their close phylogenetic relationship, they show differences in their pathogenicity and virulence mechanisms in target insects. These differences can be explored by the analysis of the pangenome, as it provides a framework for characterizing and defining the gene repertoire. Here, we report the genome of strain SC 0516. In addition, we performed the first pangenome analysis of 91 strains of Xenorhabdus and Photorhabdus, obtaining a total of 23,603 gene clusters and a core genome of 348 genes. Phylogenetic analysis performed with the core genome showed that our strain belonged to the X. nematophila group. Biological tests showed that whole cells of X. nematophila SC 0516 were more virulent than those of P. luminescens HIM3 when both were injected into Galleria mellonella larvae. In addition, we cloned and expressed the GroEL proteins of both bacteria, as this protein has been previously indicated to show insecticidal activity in the genus Xenorhabdus. Cpn60-Xn was found to be the most toxic at all concentrations tested, with an LC50 value of 102.34 ng/larva. Sequence analysis suggested that the Cpn60-Xn toxin was homologous to Cpn60-Pl; however, Cpn60-Xn contained thirty-five differentially substituted amino acid residues that could be responsible for its insecticidal activity.

PangenomeNet: a pan-genome-based network reveals functional modules on antimicrobial resistome for Escherichia coli strains

Background Discerning genes crucial to antimicrobial resistance (AMR) mechanisms is becoming more and more important to accurately and swiftly identify AMR pathogenic strains. Pangenome-wide association studies (e.g. Scoary) identified numerous putative AMR genes. However, only a tiny proportion of the putative resistance genes are annotated by AMR databases or Gene Ontology. In addition, many putative resistance genes are of unknown function (termed hypothetical proteins). An annotation tool is crucially needed in order to reveal the functional organization of the resistome and expand our knowledge of the AMR gene repertoire. Results We developed an approach (PangenomeNet) for building co-functional networks from pan-genomes to infer functions for hypothetical genes. Using Escherichia coli as an example, we demonstrated that it is possible to build co-functional network from its pan-genome using co-inheritance, domain-sharing, and protein–protein-interaction information. The investigation of the network revealed that it fits the characteristics of biological networks and can be used for functional inferences. The subgraph consisting of putative meropenem resistance genes consists of clusters of stress response genes and resistance gene acquisition pathways. Resistome subgraphs also demonstrate drug-specific AMR genes such as beta-lactamase, as well as functional roles shared among multiple classes of drugs, mostly in the stress-related pathways. Conclusions By demonstrating the idea of pan-genome-based co-functional network on the E. coli species, we showed that the network can infer functional roles of the genes, including those without functional annotations, and provides holistic views on the putative antimicrobial resistomes. We hope that the pan-genome network idea can help formulate hypothesis for targeted experimental works.

The Eco-Evo Mandala: Simplifying Bacterioplankton Complexity into Ecohealth Signatures

The microbiome emits informative signals of biological organization and environmental pressure that aid ecosystem monitoring and prediction. Are the many signals reducible to a habitat-specific portfolio that characterizes ecosystem health? Does an optimally structured microbiome imply a resilient microbiome? To answer these questions, we applied our novel Eco-Evo Mandala to bacterioplankton data from four habitats within the Great Barrier Reef, to explore how patterns in community structure, function and genetics signal habitat-specific organization and departures from theoretical optimality. The Mandala revealed communities departing from optimality in habitat-specific ways, mostly along structural and functional traits related to bacterioplankton abundance and interaction distributions (reflected by ϵ and λ as power law and exponential distribution parameters), which are not linearly associated with each other. River and reef communities were similar in their relatively low abundance and interaction disorganization (low ϵ and λ) due to their protective structured habitats. On the contrary, lagoon and estuarine inshore reefs appeared the most disorganized due to the ocean temperature and biogeochemical stress. Phylogenetic distances (D) were minimally informative in characterizing bacterioplankton organization. However, dominant populations, such as Proteobacteria, Bacteroidetes, and Cyanobacteria, were largely responsible for community patterns, being generalists with a large functional gene repertoire (high D) that increases resilience. The relative balance of these populations was found to be habitat-specific and likely related to systemic environmental stress. The position on the Mandala along the three fundamental traits, as well as fluctuations in this ecological state, conveys information about the microbiome’s health (and likely ecosystem health considering bacteria-based multitrophic dependencies) as divergence from the expected relative optimality. The Eco-Evo Mandala emphasizes how habitat and the microbiome’s interaction network topology are first- and second-order factors for ecosystem health evaluation over taxonomic species richness. Unhealthy microbiome communities and unbalanced microbes are identified not by macroecological indicators but by mapping their impact on the collective proportion and distribution of interactions, which regulates the microbiome’s ecosystem function.

Specific T Cell Receptor Gene Repertoire Profiles in Subgroups of CLL Patients with Distinct Genomic Aberrations

Chronic lymphocytic leukemia (CLL) B cells engage in multifaceted bi-directional interactions with bystander cells, including T cells. Immunogenetic studies in CLL revealed clonal expansions of T cells and shared T cell clonotypes between different patients, strongly implying clonal selection by antigens. Although the exact nature of these antigens remains largely elusive, evidence exists that the clonotypic B cell receptor immunoglobulin (BcR IG) may serve as a source of antigenic epitopes for T cells. That said, recurrent genomic aberrations associated with distinct abnormal expression profiles could represent an alternative, non mutually exclusive, source of potent immunogenic onco-antigens that might shape the T cell repertoire in CLL. On these grounds, here we interrogated the T cell receptor (TR) gene repertoire of CLL patients with different genomic aberration profiles with the aim to identify unique signatures that would allude to distinct antigen selection pressures. The study group included 46 patients with CLL, sampled before treatment initiation, who were categorized in 5 subgroups defined by a unique genomic aberration, as follows: +12, n=18; del(11q), n=10; del(13q), n=7; del(17p)/TP53mut, n=6; NOTCH1mut, n=5. Confounding effects of multiple aberrations have been minimized, as we previously established through comprehensive characterization (including FISH, SNP arrays and gene panels) that the analyzed patients carried only one of the above aberrations. Starting material was RNA extracted from blood mononuclear cells. TRBV-TRBD-TRBJ gene rearrangements were RT-PCR amplified and subjected to paired-end next generation sequencing (NGS). Raw NGS reads (n=13,213,563| median: 294,757/sample) were processed through a purpose-built bioinformatics pipeline. Only productive rearrangements (n=9,249,546 | median=199,184/sample) were taken into consideration for the computation of clonotypes i.e. TRB rearrangements with identical TRBV gene usage and amino acid complementarity-determining region 3 (CDR3) sequence. Overall, 513,984 distinct clonotypes (median=10,304 clonotypes/sample) were assessed. The main measure of clonality employed in this study was the median cumulative frequency of the 10 most expanded T cell clonotypes/sample (MCF-10). For comparisons of the clonality profiles, a group of 17 aged-matched healthy individuals were used as controls. All patients displayed oligoclonal T cell expansions with the following MCF-10 values: del(11q): 21.6%, +12: 25%, del(13q): 20.6%, NOTCH1mut: 9.1%, del(17p)/TP53mut: 12.9%; the difference between the del(11q) and +12 groups versus the NOTCH1mut group was statistically significant (p<0.05). The MCF-10 value of the control group was estimated at 17.5%, supporting the notion of age-related decrease in TR repertoire diversity. However, the del(11q), +12 and del(13q) CLL groups displayed elevated clonality, reaching statistical significance (p<0.002) in the case of +12. TRBV gene repertoire analysis revealed that the TRBV12-3 gene predominated in most groups, except for the del(17p)/TP53mut, where the predominant gene was TRBV10-3. Clonotype comparisons disclosed the presence of shared TR clonotypes both within a particular group but also between groups. Overall, 446/513,984 clonotypes were found to be shared by at least two patients across all groups; the vast majority (392/446, 88%) of shared clonotypes appeared to be CLL-biased since they did not match entries in public databases of TR clonotypes from various contexts. Subgroup-specific clonotypes were identified for all aberrations examined; these emerged as unique to the particular subgroups, as revealed by extensive comparisons against both public databases but also a large TR clonotype database from CLL available to us from our previous studies. In conclusion, recurrent genomic aberrations, especially large chromosomal abnormalities, display an oligoclonal TR gene repertoire. The distinct immunogenetic profile of each group examined here and, most importantly, the existence of subgroup-specific clonotypes, suggest that abnormal protein expression and gene dosage effects likely represent a relevant source of CLL-specific selecting antigens. Disclosures Scarfo: Janssen: Honoraria, Other: Travel grants; Astra Zeneca: Honoraria; Abbvie: Honoraria. Anagnostopoulos: Abbvie: Other: clinical trials; Sanofi: Other: clinical trials ; Ocopeptides: Other: clinical trials ; GSK: Other: clinical trials; Incyte: Other: clinical trials ; Takeda: Other: clinical trials ; Amgen: Other: clinical trials ; Janssen: Other: clinical trials; novartis: Other: clinical trials; Celgene: Other: clinical trials; Roche: Other: clinical trials; Astellas: Other: clinical trials . Ghia: AbbVie: Consultancy, Honoraria, Research Funding; Acerta/AstraZeneca: Consultancy, Honoraria, Research Funding; AstraZeneca: Consultancy, Honoraria, Research Funding; ArQule/MSD: Consultancy, Honoraria; BeiGene: Consultancy, Honoraria; Celgene/Juno/BMS: Consultancy, Honoraria; Gilead: Consultancy, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Roche: Consultancy, Honoraria; Sunesis: Research Funding. Haferlach: MLL Munich Leukemia Laboratory: Other: Part ownership. Rosenquist: Roche: Honoraria; Janssen: Honoraria; Illumina: Honoraria; AstraZeneca: Honoraria; Abbvie: Honoraria. Stamatopoulos: Gilead: Honoraria, Research Funding; Abbvie: Honoraria, Research Funding; AstraZeneca: Honoraria, Research Funding; Janssen: Honoraria, Research Funding. Baliakas: Janssen: Honoraria; Gilead: Honoraria, Research Funding; Abbvie: Honoraria. Chatzidimitriou: Abbvie: Honoraria, Research Funding; Janssen: Honoraria, Research Funding.

Distinct Modes of Ongoing Antigen Interactions Shape Intraclonal Dynamics in Splenic Marginal Zone Lymphoma

Almost one-third of all splenic marginal zone lymphoma (SMZL) cases express B cell receptor immunoglobulin (BcR IG) encoded by the IGHV1-2*04 gene. Such cases display a distinctive profile of genomic aberrations (e.g. higher incidence of NOTCH2 and KLF2 mutations) and a more aggressive clinical course compared to SMZL cases utilizing other IGHV genes. Such skewing of the BcR IG gene repertoire implicates antigen selection in SMZL ontogeny. Although the supportive evidence is compelling, it mostly derives from low-throughput approaches, which are inherently limited in their capacity to capture the complexity of the BcR IG gene repertoire. This hinders the comprehensive assessment of the subclonal architecture of SMZL that could offer insight into the dynamics of antigen-IG interactions. Here, we sought to overcome this limitation through a high-throughput immunogenetic investigation of SMZL, focusing on the detailed characterization of somatic hypermutation (SHM) and intraclonal diversification (ID) profiles. Our study included 22 cases utilizing the IGHV1-2*04 gene and 36 cases utilizing other IGHV genes. IGHV-IGHD-IGHJ (IGH) gene rearrangements were PCR-amplified and libraries were sequenced on the Illumina MiSeq platform. Data was analyzed with the IMGT/HighV-QUEST and TRIP software as well as a novel bioinformatics/biostatistics pipeline. Clonotypes were defined as unique combinations of a given IGHV gene+VH CDR3 amino acid (aa) sequence. Only IGH gene rearrangement sequences assigned to the dominant clonotypes of each case were assessed. In detail, all nucleotide variants (nt vars, i.e. all sequences clustered in the same dominant clonotype yet displaying distinct SHM profiles) were identified and further analyzed. Starting from the most abundant nt var, a network was built representing its connections with all other nt vars. For this analysis, we introduce the terms 'most relevant pathway' (MRP) corresponding to the pathway including connected nt vars with the highest total number of IGH sequences; and 'longest mutational pathways' (LMP) corresponding to the pathways with the highest number of nt vars (Fig. 1). Different graph metrics assessed the impact of ID in different SMZL subgroups: the first one focuses on the 'most relevant pathway' and quantifies SHM convergence [ratio of the total number of IGH sequences corresponding to the nt vars of this pathway to the number of IGH sequences in the most abundant nt var]; while the second refers to the length of the 'longest mutational pathways'. Cases lacking additional connected nt vars [length of the LMP=1; 3 IGHV1-2*04 cases and 4 non-IGHV1-2*04 cases] were excluded. Consequently, the analysis included 19 IGHV1-2*04 cases and 32 non-IGHV1-2*04 cases. Significant differences were noted in the SHM and ID profiles between groups; the IGHV1-2*04 group had significantly (p<0.01) higher convergence values ranging from 0.009 to 1.243 (median: 0.102), as opposed to the non-IGHV1-2*04 group (range: 0.002-1.13, median: 0.014), overall suggesting that stronger selective pressures act in SMZL cases expressing the IGHV1-2*04 versus others. Moreover, IGHV1-2*04 cases displayed significantly (p<0.01) longer mutational pathways (length range: 2-6, median: 3) compared to the other group (range: 2-5, median: 2), alluding to more pronounced ID arising due to ongoing SHM. Finally, all mutations leading to aa changes were analyzed in the context of ID networks. More recurrent aa mutations were identified amongst cases with higher levels of convergence. For instance, the VH FR2 M39I change, one of the most prominent recurrent SHMs in the IGHV1-2*04 group, was found in the most abundant nt var in 13/19 IGHV1-2*04 cases, while it was identified in nt vars with extra mutations in another 3 cases. Of interest, it was present at the end of the mutational pathways in these 16 cases, whilst in the other group it was present only in one case using the IGHV1-2*02 gene, and absent in the rest (p<0.01). In conclusion, in the first large-scale high-throughput immunogenetic analysis of SMZL we provide strong evidence for more pronounced antigenic pressure in cases utilizing IGHV1-2*04 versus other IGHV genes. Our findings highlight a unique subclonal architecture for IGHV1-2*04 SMZL and corroborate the hypothesis that this group may represent a distinct molecular variant of SMZL. Figure 1 Figure 1. Disclosures Rossi: Abbvie: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; AstraZeneca: Honoraria, Research Funding; Gilead: Honoraria, Research Funding; Verastem: Honoraria, Research Funding; Roche: Honoraria, Research Funding; Cellestia: Honoraria, Research Funding. Chatzidimitriou: Janssen: Honoraria, Research Funding; Abbvie: Honoraria, Research Funding. Stamatopoulos: Janssen: Honoraria, Research Funding; Gilead: Honoraria, Research Funding; Abbvie: Honoraria, Research Funding; AstraZeneca: Honoraria, Research Funding.