Biology subjects

Kupczok, A.

Publications and source records attributed to Kupczok, A..

At least 19 recordsLinked to original sources

bioRxiv2026

RdRpCATCH: A unified resource for RNA virus discovery using viral RNA-dependent RNA polymerase profile Hidden Markov models

Recent advances in large-scale sequence mining have expanded our knowledge of RNA virus diversity. Most genome mining approaches for detecting RNA viruses that encode RNA-dependent RNA polymerase (RdRp) rely on identifying this conserved protein by employing profile Hidden Markov Models (pHMMs) to scan sequencing datasets. Recently, several new pHMM databases for RdRp detection have been released, each following distinct design principles. However, their relative performance is unclear and their accessibility to users without specialized computational expertise is limited. Here, we introduce the RdRp Collaborative Analysis Tool with Collections of pHMMs (RdRpCATCH: https://github.com/dimitris-karapliafis/RdRpCATCH), developed to consolidate publicly available RdRp pHMM resources into a single, accessible platform. RdRpCATCH enables the scanning of (meta)transcriptomic assemblies to discover RNA viruses and provides subsequent taxonomic annotation of detected contigs. A comparative analysis of RdRp pHMM databases reveals that most are highly effective at detecting known diversity of RNA viruses while minimizing false positives, supporting their joint use within RdRpCATCH. RdRpCATCH is distributed as both a conda package and a web server application (https://rdrpcatch.bioinformatics.nl), facilitating access for researchers with diverse expertise. By integrating multiple pHMM resources, this unified framework addresses fragmentation in the field and reduces technical barriers, enabling comprehensive viral discovery.

bioinformatics↗

bioRxiv2026

Phylogenetic support of pebS as a phage-exclusive auxiliary metabolic gene

Marine picocyanobacteria, including the genera Prochlorococcus and Synechococcus, are major contributors to oceanic photosynthesis and global primary production. Their populations are influenced by T4-like cyanophages, which frequently encode auxiliary metabolic genes (AMGs) capable of altering host metabolism during infection. One such AMG, pebS, encodes a ferredoxin-dependent bilin reductase (FDBR) phycoerythrobilin (PEB) synthase, which converts biliverdin IX to PEB. In contrast, cyanobacteria perform a two-step reaction using the FDBR enzymes PebA (15,16-dihydrobiliverdin:ferredoxin oxidoreductase) and PebB (PEB:ferredoxin oxidoreductase), whereas pebS has not been reported in cyanobacterial genomes. Here, we re-evaluated whether pebS is truly restricted to cyanophages by searching the Ocean Gene Atlas and all available cyanobacterial genomes at NCBI using a cyanophage-derived PebS sequence as query. Using protein phylogenies, we find that most search hits group with PebA or PebB, while few sequences from cyanobacterial genome assemblies were confirmed to belong to PebS based on phylogenetic placement. However, genomic context analysis of these pebS sequences revealed that they are phage-derived, consistent with cyanophage infection at the time of sampling. In conclusion, our results support that pebS is absent in cyanobacterial genomes, raising questions about the evolutionary and biochemical rationale for the two-step reduction of biliverdin IX to PEB in these organisms.

microbiology↗

bioRxiv2026

Identification and Classification of Expressed Orphan Genes, Spurious Orphan Genes, and Conserved Genes in the Human Gut Microbiome

Orphan genes - genes lacking detectable homologs outside a species - are widespread in microbial genomes and are thought to contribute to their adaptation and molecular innovation. However, not all predicted orphan genes may represent novel functional coding sequences. False positive orphan genes, also called spurious orphan genes, can arise from gene prediction errors. We reason that orphan genes lacking detectable expression are more likely to be spurious. To test this, we combined large-scale metatranscriptomic profiling of the human gut microbiome with machine learning to distinguish expressed orphan genes from spurious ones and to compare them with conserved genes found in multiple species. Using nearly 5,000 metatranscriptome libraries, we identified [~]218,000 orphan genes supported by expression evidence, while [~]330,000 predicted orphan genes lacked detectable expression, and were classified as spurious. We extracted 154 features for sequence, structural, and evolutionary properties for each gene and trained XGBoost classifiers while accounting for genomic representation. The models achieved an area under the receiver operating characteristic curve (AUC) of 0.82 in distinguishing expressed orphan genes from spurious orphan genes and an AUC of 0.93 in distinguishing expressed orphan genes from conserved genes. SHAP-based interpretation revealed clear biological signals. Particularly, expressed orphans were present in more genomes than spurious ones and expressed orphan genes were shorter than conserved genes. This work improves orphan gene discovery and suggests that expressed orphan genes differ systematically from conserved genes and spurious orphan genes in sequence composition, structural constraints, and evolutionary signals. Significance statementSpecies-specific genes, also called orphan genes, are abundant in bacterial genomes and have the potential to drive evolutionary innovation. However, the study of orphan genes is confounded by annotation errors that produce spurious gene predictions. Here, we leveraged nearly 5,000 metatranscriptomes from the human gut microbiome and implemented machine learning models to distinguish expressed orphan genes from non-expressed, likely spurious, genes and conserved genes. We show that a substantial fraction of predicted orphan genes lacks expression support and they exhibit distinct sequence and evolutionary signatures from expressed orphans. By integrating expression evidence with sequence-, structure-, and evolution-based features, our work refines orphan gene catalogs. This work improves the reliability of orphan gene discovery and provides new insights into the biological characteristics of orphan genes in human microbial communities.

genomics↗

bioRxiv2025

Microdiversity is higher in temperate than in virulent bacteriophages from soil environments

Bacteriophages are not only the most ubiquitous biological entity on earth, they also display remarkable genetic diversity across and within populations. While macrodiversity has been extensively studied, the drivers of microdiversity (intra-species genetic diversity) remain poorly understood, particularly in relation to phage lifestyle. The distinguishing ability of temperate phages to integrate themselves into the host genome has an unknown influence on the microdiversity present. This difference in microdiversity could impact the adaptability of phages to (a)biotic factors. To identify a possible association between microdiversity and lifestyle, we analysed 12 existing viromics datasets focusing on soil bacteriophages, including 43 171 viral genomes in total. We found that phages predicted to be temperate consistently exhibit higher microdiversity than their virulent counterparts in eight of 12 datasets. Of the remaining four datasets, one showed the opposite trend and three datasets did not show a significant trend at all. The detected pattern holds across multiple quality thresholds and lifestyle prediction methods. These findings suggest that lifestyle has a significant impact on genetic variation within phage populations, with potential implications for phage-host coevolution and environmental adaptability.

genomics↗

bioRxiv2025

The evolutionary tale of tailocins in Pectobacterium show genus-wide homologous recombination and inversions of tail fiber genes

Due to their ability to kill closely related strains, phage tail-like bacteriocins, also called tailocins, play an important role in shaping bacterial communities. One such tailocin, called carotovoricin, is also known to be present in the Pectobacterium genus. However, little is known about its evolutionary dynamics and the scope of impact on species interactions in this genus. To investigate the diversity and evolution of carotovoricin, we performed a genus-wide, phylogenetically-structured pangenome study. This analysis inferred that the gene cluster responsible for carotovoricin biosynthesis is conserved across the genus and is located in the same gene neighborhood in all the species. Within the carotovoricin cluster, the tail fiber genes, which determine the host range specificity, exhibit high variability and discordance with the species phylogeny. We show evidence for an evolutionary mechanism involving recombination-mediated exchange of these tail fiber loci across the entire Pectobacterium genus, which complements the previously known mechanism for DNA sequence inversion to maintain tailocin polymorphism at the population level. In addition, the ability to exchange tail-fiber loci in a highly targeted and genus-wide manner could influence the community dynamics in nutrient rich environments such as infected plant tissues. In conclusion, the strong signal for carotovoricin retention and ability to exchange tail fibers indicates that it significantly contributes to the community interactions of the Pectobacterium phytopathogens. Significance StatementA widespread presence of tailocins among various gram-negative bacteria and maintenance of their tail fiber diversity underscore their role in inter-bacterial interactions. A tailocin is also found to be conserved in Pectobacterium, a pathogen causing soft rot. However, the mechanism maintaining the diversity of the tailocin tail fibers, which enable recognition of the target bacteria, is not yet completely understood. Here, we characterized the genomic diversity of this tailocin and discovered that the diversity is maintained through the exchange of the tail fiber locus DNA across the genus.

evolutionary biology↗

bioRxiv2025

Machine learning can identify orphans that have diverged into the ''twilight zone'' of sequence similarity

Species-specific orphan genes (orphans) lack homologues outside of a given taxon and frequently underlie unique species traits. It is thus important to elucidate the evolutionary origins of orphans. Orphan genes can result from sequence divergence beyond recognition, when homologous proteins diverge to an extent at which tools that rely on sequence similarity to establish homology can no longer identify them as homologues. Orphans can also result from other processes, including de novo gene emergence from previously noncoding sequences, in which a homologous protein-coding gene truly does not exist. Here we propose that orphans resulting from divergence might be recognizable from their patterns of non-statistically significant similarity hits which are almost always discarded. To test this, we simulated diverged orphan protein sequences based on conserved proteins from the Unified Human Gastrointestinal Protein catalogue (UHGP) and used reversed protein sequences as negative data sets. We trained four machine learning classifiers on features extracted from the similarity search tool DIAMONDs output, like total query coverage or maximum bit score. We tested the influence of evolutionary parameters such as simulation tree branch length, indel rate and among-site rate heterogeneity. We found that the performance of the models depends on the simulation parameters: when the underlying simulated divergence was moderate, accuracy reached [~]90%, but when extremely diverged scenarios where simulated accuracy dropped to [~]70%. The most important features for the classification were the number of alignments (hits) and the minimum hit E-value. When applying our classifier on a set of [~]170,000 eligible simulated orphans from the UHGP dataset, we found that [~]30% of them are predicted to be divergent and these are shorter and more disordered than the rest. Our classifiers and pre-processing python scripts are available at https://github.com/emiliostassios/Classification-of-divergent-genes-using-ML and can be readily used as a computationally fast means to obtain a candidate set of diverged orphans from any similarity search output. Therefore, our work allows to study such orphans across the tree of life and in doing so to recover cases of remote homology, get better estimates of the evolutionary age of protein families, detect cases of rapid divergence and more generally better understand how genetic novelty arises.

bioinformatics↗

bioRxiv2025

Transcriptome mining reveals diversity and evolution of iflaviruses in Lepidoptera

Insects are associated with a wide variety of diverse RNA viruses, including iflaviruses, a group of positive stranded RNA viruses that mainly infect arthropods. Whereas some iflaviruses cause severe diseases in insects, numerous iflaviruses that were detected in healthy populations of butterflies and moths (order: Lepidoptera) do not show apparent symptoms. Compared to other hosts, only few iflavirus genomes for lepidopteran hosts could be found in publicly available databases and we know little about the occurrence of iflaviruses in natural and laboratory lepidopteran populations. To expand the known diversity of iflaviruses in Lepidoptera, we developed a pipeline to automatically reconstruct virus genomes from public transcriptome data. We reconstructed 1,548 virus genomes from 55 different lepidopteran species that we were identified as coding-complete based on their length. To include incompletely assembled genomes, we developed a reference-based patching approach, resulting in 240 patched genomes. By including publicly available genomes, we inferred a phylogeny consisting of 139 non-redundant iflavirus genomes. Of these, 65 represent novel complete genomes, of which 39 might even belong to novel virus species. Our analysis expanded virus host range, where highly similar viruses were found in the transcriptomes of different lepidopteran species, genera or even families. Additionally, we find two groups of lepidopteran species depending on the diversity of viruses that infect them: some species were only infected by closely related viruses, whereas other species are infected by highly diverse viruses from different regions of the phylogeny. Finally, we show that the evolution of one virus species, Iflavirus betaspexiguae, is impacted by recombination within the species, which is also supported by the co-occurrence of multiple strains within data sets. Our analysis demonstrates how data mining of publicly available sequencing data can be used at a large scale to reconstruct intra-family viral diversity which serves as a basis to study virus host range and evolution. Our results contain numerous novel viruses and novel virus-host associations, including viruses for relevant insect pests, highlighting the impact of iflaviruses in insect ecology and as potential biological control agents in the future.

genomics↗

bioRxiv2024

Pangenomics to understand prophage dynamics in the Pectobacterium genus and the radiating lineages of P. brasiliense

Bacterial pathogens of the genus Pectobacterium are responsible for soft rot and blackleg disease in a wide range of crops and have a global impact on food production. The emergence of new lineages and their competitive succession is frequently observed in Pectobacterium species, in particular in P. brasiliense. With a focus on one such recently emerged P. brasiliense lineage in the Netherlands that causes blackleg in potatoes, we studied genome evolution in this genus using a reference-free graph-based pangenome approach. We clustered 1,977,865 proteins from 454 Pectobacterium spp. genomes into 30,156 homology groups. The Pectobacterium genus pangenome is open and its growth is mainly contributed by the accessory genome. Bacteriophage genes were enriched in the accessory genome and contributed 16% of the pangenome. Blackleg-causing P. brasiliense isolates had increased genome size with high levels of prophage integration. To study the diversity and dynamics of these prophages across the pangenome, we developed an approach to trace prophages across genomes using pangenome homology group signatures. We identified lineage-specific as well as generalist bacteriophages infecting Pectobacterium species. Our results capture the ongoing dynamics of mobile genetic elements, even in the clonal lineages. The observed lineage-specific prophage dynamics provide mechanistic insights into Pectobacterium pangenome growth and contribution to the radiating lineages of P. brasiliense.

bioinformatics↗

bioRxiv2024

Goldfinder: Unraveling Networks of Gene Co-occurrence and Avoidance in Bacterial Pangenomes

The pangenome is the set of all genes present in a prokaryotic species. Most pangenomes contain many accessory genes that are present in only some of the species members. Genes need to function together, and it has been suggested that selection for certain gene combinations affects the structure of prokaryotic pangenomes. Nevertheless, genes might also co-occur simply due to being linked on the genome, and efficient tools are needed to distinguish linkage from co-selection. Here we present Goldfinder, an approach to infer co-occurrence and co-avoidance between gene pairs by taking the phylogenetic relationships of the species into account. The approach is implemented in an efficient Python script available at https://github.com/fbaumdicker/goldfinder. We also provide scripts for clustering co-occurring genes and for visualizing the resulting co-occurrence and co-avoidance networks in Cytoscape. In comparison to the co-occurrence inference tool Coinfinder, Goldfinder finds fewer co-occurring pairs in a real species pangenome, suggesting that fewer spurious associations due to phylogenetic dependencies are detected. We conclude that Goldfinder is a fast and accurate tool to infer gene co-occurrence and co-avoidance, which will enable large-scale analyses to infer co-selected genes across bacterial pangenomes.

bioinformatics↗

bioRxiv2023

Virus-encoded Shemin pathway highlights the importance of tetrapyrrole metabolism during host infection

Tetrapyrroles such as heme, chlorophyll, or vitamin B12 are among the most complex molecules synthesised by nature and play a crucial role in many vital processes such as respiration, photosynthesis, methanogenesis, and catalysis. All modified tetrapyrroles are derived from the common precursor molecule 5-aminolevulinic acid (5-ALA). However, 5-ALA can be synthesised in two different ways. While alphaproteobacteria, mammals, and birds use the so-called Shemin pathway, all other bacteria, archaea, and plants use the C5 pathway. Here, we present evidence for the presence of 5-ALA synthase genes of the Shemin pathway in the genomes of viruses (valaS) that infect bacteria (bacteriophages) in marine and freshwater environments. These genes either occur in a three-gene cassette with two heme catabolising enzymes involved in linear tetrapyrrole biosynthesis, as broken cassettes or as individual genes. valaS encodes a functional enzyme, as shown by the enzymatic turnover of the co-substrates succinyl-Coenzyme A and glycine in an Escherichia coli strain overexpressing valaS. Phylogenetic analysis in combination with three-dimensional structure prediction supported our data that the viral sequences encode active enzymes. Interestingly, the viral valaS sequence is capable of functionally complementing a 5-ALA-auxotrophic Escherichia coli strain, which lacks one of the two enzymes of the C5 pathway. Host prediction suggests that different valaS carrying phages might infect bacteria that either utilise the Shemin or the C5 pathway for 5-ALA biosynthesis. Our results thus support the hypothesis that metabolic reprogramming and maintenance of the host bacteriums energy metabolism are crucial during bacteriophage infection and that tetrapyrroles play a key role in this process.

microbiology↗

bioRxiv2023

Large-scale investigation of orphan genes in the human gut microbiome elucidates their evolutionary origins

Species-specific genes, also known as orphans, are ubiquitous across lifes domains. In prokaryotes, species-specific orphan genes (SSOGs) are mostly thought to originate in external elements such as viruses, followed by horizontal gene transfer, while the scenario of native origination, through rapid divergence or de novo, is mostly dismissed. However, quantitative evidence supporting either scenario is lacking. Here, we systematically analyzed genomes from 4,644 human gut microbiome species and identified over 600,000 unique SSOGs, representing an average of 2.6% of a given species pangenome. These sequences are mostly rare within each species yet show signs of purifying selection. Overall, SSOGs use optimal codons less frequently, and their proteins are more disordered than those of conserved genes (i.e., non-SSOGs). Importantly, across species, the GC content of SSOGs closely matches that of conserved ones. In contrast, the [~]5% of SSOGs that share similarity to known viral sequences have distinct characteristics, including lower GC content. Thus, SSOGs with similarity to viruses differ from the remaining SSOGs, contrasting an external origination scenario for most SSOGs. By examining the orthologous genomic region in closely related species, we show that a small subset of SSOGs likely evolved natively de novo and find that these genes also differ in their properties from the remaining SSOGs. Our results challenge the notion that external elements are the dominant source of prokaryotic genetic novelty and will enable future studies into the biological role and relevance of species-specific genes in the human gut.

evolutionary biology↗

bioRxiv2022

Arabidopsis latent virus 1, a comovirus widely spread in Arabidopsis thaliana collections

O_LITranscriptome studies of Illumina RNA-seq datasets of different Arabidopsis thaliana natural accessions and T-DNA mutants revealed the presence of two virus-like RNA sequences which showed the typical two segmented genome characteristics of a comovirus. C_LIO_LIThis comovirus did not induce any visible symptoms in infected Arabidopsis plants cultivated under standard laboratory conditions. Hence it was named Arabidopsis latent virus 1 (ArLV1). Virus infectivity in Arabidopsis plants was confirmed by RT-qPCR, transmission electron microscopy and mechanical inoculation. ArLV1 can also mechanically infect Nicotiana benthamiana, causing distinct mosaic symptoms. C_LIO_LIA bioinformatics investigation of Arabidopsis RNA-Seq repositories, including nearly 6500 Sequence Read Archives (SRAs) in the NCBI SRA database, revealed the presence of ArLV1 in 25% of all archived natural Arabidopsis accessions and in 8.5% of all analyzed SRAs. ArLV1 could also be detected in Arabidopsis plants collected from the wild. C_LIO_LIArLV1 is highly seed-transmissible with up to 40% incidence on the progeny derived from infected Arabidopsis plants. This has likely led to a worldwide distribution in the model plant Arabidopsis with yet unknown effects on plant performance in a substantial number of studies. C_LI Plain language summaryWe identified Arabidopsis latent virus 1 (ArLV1), a comovirus that infects the model plant Arabidopsis thaliana without causing any visible symptoms. It is efficiently spread by transmission via seeds to the plant progeny. ArLV1 is infectious to Arabidopsis plants and another model plant, Nicotiana benthamiana. By analyzing public sequencing data, we found that ArLV1 is widely spread in Arabidopsis laboratory collections worldwide. Moreover, it was also detected in wild Arabidopsis plants collected from different locations in the Netherlands and Spain, suggesting that it is a virus that naturally occurs in Arabidopsis.

plant biology↗

bioRxiv2022

Co-transfer of functionally interdependent genes contributes to genome mosaicism in lambdoid phages

Lambdoid (or Lambda-like) phages, are a group of related temperate phages that can infect Escherichia coli and other gut bacteria. A key characteristic of these phages is their mosaic genome structure which served as basis for the "modular genome hypothesis". Accordingly, lambdoid phages evolve by transferring genomic regions, each of which constitutes a functional unit. Nevertheless, it is unknown which genes are preferentially transferred together and what drives such co-transfer events. Here we aim to characterize genome modularity by studying co-transfer of genes among 95 distantly related lambdoid (pro-)phages. Based on gene content, we observed that the genomes cluster into twelve groups, which are characterized by a highly similar gene content within the groups and highly divergent gene content across groups. Highly similar proteins can occur in genomes of different groups, indicating that they have been transferred. About 26% of homologous protein clusters in the four known operons (i.e., the early left, early right, immunity, and late operon) engage in gene transfer, which affects all operons to a similar extent. We identified pairs of genes that are frequently co-transferred and observed that these pairs tend to be in close proximity to one another on the genome. We find that frequently co-transferred genes are involved in related functions and highlight interesting examples involving structural proteins, the CI repressor and Cro regulator, proteins interacting with DNA, and membrane-interacting proteins. We conclude that epistatic effects, where the functioning of one protein depends on the presence of another, plays an important role in the evolution of the modular structure of these genomes. Data summaryThe genomes used in this research are publicly available (Table S1). All supporting data is available in supplementary tables. Source code and documentation to calculate GRR is available under GPLv2 (https://github.com/annecmg/GRRpair). Impact statementTemperate phages, viruses that can integrate their own genetic material into bacterial genomes, are pervasive mobile genetic elements that can influence bacterial fitness in manifold ways. The E. coli phage lambda has been a model phage of molecular biology for decades. Lambdoid phages are highly prevalent in Enterobacteria such as E. coli and Salmonella, have a mosaic-like genome, the same genome architecture as lambda, and can recombine with phage lambda. Nevertheless, these phages can be very distinct, and no lambdoid core genome exits. Although lambdoid phage genomes have been studied for decades, we know relatively little about how they evolve. Early observations led to the modular genome hypothesis, according to which, phages are assemblages of genetic modules. But what determines the structure of these modules and which genes do preferentially occur together in modules? In this study, we provide answers to these questions using a novel computational approach that allows to infer gene transfer events between distantly related phages despite the absence of a core genome. We find that co-transfer of functionally related genes is frequent during the evolution of lambdoid phages. This suggests epistatic interactions among these genes, i.e., the co-transferred genes likely need to function together to ensure a viable phage. A prime example is the co-transfer of structural genes, such as genes encoding for the phage capsid or the phage tail. Additionally, we also find co-transfer of known interacting regulatory genes and co-transfer between functionally related genes that have so far been unknown to interact. Together, our analysis provides novel insights into the evolution of temperate phages. Moreover, our approach, which allows to identify gene transfer in the absence of a core phylogeny might be valuable for studying the evolution of other fast-evolving genomes, including viruses of other hosts.

microbiology↗

bioRxiv2022

Fixation dynamics of beneficial alleles in prokaryotic polyploid chromosomes and plasmids

Theoretical population genetics has been mostly developed for sexually reproducing diploid and for monoploid (haploid) organisms, focusing on eukaryotes. The evolution of bacteria and archaea is often studied by models for the allele dynamics in monoploid populations. However, many prokaryotic organisms harbor multicopy replicons - chromosomes and plasmids - and theory for the allele dynamics in populations of polyploid prokaryotes remains lacking. Here we present a population genetics model for replicons with multiple copies in the cell. Using this model, we characterize the fixation process of a dominant beneficial mutation at two levels: the phenotype and the genotype. Our results show that, depending on the mode of replication and segregation, the fixation time of mutant phenotypes may precede the genotypic fixation time by many generations; we term this time interval the heterozygosity window. We furthermore derive concise analytical expressions for the occurrence and length of the heterozygosity window, showing that it emerges if the copy number is high and selection strong. Replicon ploidy thus allows for the maintenance of genetic variation following phenotypic adaptation and consequently for reversibility in adaptation to fluctuating environmental conditions.

evolutionary biology↗

bioRxiv2021

Disentangling the genetic basis of rhizosphere microbiome assembly in tomato

Microbiomes play a pivotal role in plant growth and health, but the genetic factors involved in microbiome assembly remain largely elusive. Here, 16S amplicon and metagenomic features of the rhizosphere microbiome were mapped as quantitative traits of a recombinant inbred line population of a cross between wild and domesticated tomato. Gene content analysis of prioritized tomato QTLs suggested a genetic basis for differential recruitment of various rhizobacterial lineages, including a Streptomyces-associated 6.31-Mbp region harboring tomato domestication sweeps and encoding, among others, the iron regulator FIT and the aquaporin SlTIP2.3. Within metagenome-assembled genomes of the rhizobacterial lineages Streptomyces and Cellvibrio, we identified microbial genes involved in metabolism of plant polysaccharides, iron, sulfur, trehalose, and vitamins, whose genetic variation associated with either modern or wild tomato QTLs. Integrating microbiomics and quantitative plant genetics pinpointed putative plant and reciprocal microbial traits underlying microbiome assembly, thereby providing the first step towards plant-microbiome breeding programs.

microbiology↗

bioRxiv2021

Pangenome evolution in environmentally transmitted symbionts of deep-sea mussels is governed by vertical inheritance

Microbial pangenomes vary across species; their size and structure are determined by genetic diversity within the population and by gene loss and horizontal gene transfer (HGT). Many bacteria are associated with eukaryotic hosts where the host colonization dynamics may impact bacterial genome evolution. Host-associated lifestyle has been recognized as a barrier to HGT in parentally transmitted bacteria. However, pangenome evolution of environmentally acquired symbionts remains understudied, often due to limitations in symbiont cultivation. Using high-resolution metagenomics, here we study pangenome evolution of two co-occurring endosymbionts inhabiting Bathymodiolus brooksi mussels from a single cold seep. The symbionts, sulfur-oxidizing (SOX) and methane-oxidizing (MOX) gamma-proteobacteria, are environmentally acquired at an early developmental stage and individual mussels may harbor multiple strains of each species. We found differences in the accessory gene content of both symbionts across individual mussels, which are reflected by differences in symbiont strain composition. Compared to core genes, accessory genes are enriched in functions involved in genome integrity. We found no evidence for recent horizontal gene transfer between both symbionts. A comparison between the symbiont pangenomes revealed that the MOX population is less diverged and contains fewer accessory genes, supporting that the MOX association with B. brooksi is more recent than that of SOX. Our results show that the pangenomes of both symbionts evolved mainly by vertical inheritance. We conclude that environmentally transmitted symbionts that associate with individual hosts over their lifetime show features of narrow symbioses, where the frequency of HGT is constrained.

evolutionary biology↗

bioRxiv2020

Insertion and deletion evolution reflects antibiotics selection pressure in a Mycobacterium tuberculosis outbreak.

In genome evolution, genetic variants are the source of diversity, which natural selection acts upon. Treatment of human tuberculosis (TB) induces a strong selection pressure for the emergence of antibiotic resistance in the infecting Mycobacterium tuberculosis (MTB) strains. MTB evolution in response to treatment has been intensively studied and mainly attributed to point substitutions. However, the contribution of insertions and deletions (indels) to MTB genome evolution remains poorly understood. Here, we analyzed a multi-drug resistant MTB outbreak for the presence of high-quality indels and substitutions. We find that indels are significantly enriched in genes conferring antibiotic resistance. Furthermore, we show that indels are inherited during the outbreak and follow a molecular clock with an evolutionary rate of 5.37e-9 indels/site/year, which is 23x lower compared to the substitution rate. Inherited indels may co-occur with substitutions in genes along related biological pathways; examples are iron storage and resistance to second-line antibiotics. This suggests that epistatic interactions between indels and substitutions affect antibiotic resistance and compensatory evolution in MTB. Author summaryMycobacterium tuberculosis (MTB) is a human pathogen causing millions of deaths every year. Its genome evolution has been intensively characterized through point substitutions, i.e., nucleotide exchanges that are inherited. Additional mutations are short or long insertions and deletions of nucleotides, termed indels. Short indels in genes might change the reading frame and disrupt the gene product. Here we show that antibiotic treatment has a strong impact on indel evolution in an MTB outbreak. Namely, indels occur frequently in genes causing antibiotic resistance upon disruption. Furthermore, we show that the molecular clock, i.e., the temporal emergence of variants over time, holds for short indels in MTB genomes. Finally, we observe that indels may co-occur with substitutions in genes along related biological pathways. These results support the notion that indels are important contributors to MTB evolution. We anticipate that including indels in the analyses of MTB outbreaks will improve our understanding of antibiotic resistance evolution.

microbiology↗

bioRxiv2019

A symbiont phage protein aids in eukaryote immune evasion

Phages are increasingly recognized as important members of host associated microbial communities. While recent studies have revealed vast genomic diversity in the virosphere, the new frontier is to understand how newly discovered phages may affect higher order processes, such as in the context of host-microbe interactions. Here, we aim to understand the tripartite interplay between phages, bacterial symbionts and marine sponges. In a viromics approach, we discover 491 novel viral clusters and show that sponges, as filter-feeding organisms, are distinct viral niches. By using a nested sampling design, we show that each sponge individual of the four species investigated harbours its own unique virome, regardless of the tissue investigated. We further discover a novel, symbiont phage-encoded ankyrin domain-containing protein which appears to be widely spread in phages of many host-associated contexts including human. The ankyrin protein (ANKp) modulates the eukaryotic immune response against bacteria as confirmed in macrophage infection assays. We predict that the role of ANKp in nature is to facilitate co-existence in the tripartite interplay between phages, symbionts and sponges and possibly in many other host-microbe associations.

microbiology↗