Biology subjects

Dagan, T.

Publications and source records attributed to Dagan, T..

At least 19 recordsLinked to original sources

bioRxiv2026

Regulatory and metabolic integration accompany plasmid domestication during adaptation to a plant-associated lifestyle

Plasmids are major drivers of evolutionary innovation and ecological adaptation in prokaryotes. Plasmids that confer beneficial functions on their host may become domesticated through long-term coevolution, resulting in stable vertical inheritance within a bacterial lineage. How this process contributes to the evolution of bacteria associated with eukaryotic hosts remains poorly understood. Here, we investigate the function of the two large domesticated plasmids, LPP1 and LPP2, in Pantoea agglomerans associated with wheat. We show that loss of LPP1 reduces bacterial fitness during wheat colonization and impairs the utilization of diverse carbon sources. Comparative transcriptomics further reveal that curing of either plasmid extensively remodels chromosomal gene expression under both laboratory and wheat-associated conditions, whereas genome-scale metabolic reconstruction predicts that many metabolic pathways are jointly encoded by plasmid and chromosome. This functional integration extends beyond plasmid-specific pathways, as plasmid curing disrupts the utilization of substrates metabolized exclusively by chromosomally-encoded enzymes. Our results indicate that LPP1 promotes P. agglomerans association with wheat primarily through its regulatory and metabolic integration with the chromosome rather than through the simple addition of accessory genes. We propose that LPP1, and likely LPP2, represent chromosome-like symbiosis plasmids whose domestication reflects long-term selection for plant-associated lifestyle in Pantoea. Our findings suggest that persistent selection imposed by symbiotic lifestyles drives plasmid domestication, progressively transforming mobile plasmids into chromosome-like replicons that are integral components of bacterial genomes.

microbiology↗

bioRxiv2026

Horizontal transfer promotes allele segregation in multicopy plasmids

Plasmids reside within prokaryotic cells in multiple copies and can spread horizontally between hosts. Their multicopy nature enables intracellular allele diversity (heteroplasmy), and their segregation depends on the modes of plasmid replication and partition. Horizontal plasmid transfer has the potential to alter plasmid allele composition, however its impact on plasmid allele dynamics remains poorly understood. Here, we show that conjugative plasmid transfer accelerates the segregation of plasmid heteroplasmy by promoting the emergence of homoplasmic hosts. Using a quantitative experimental system to track plasmid allele dynamics in Acinetobacter baylyi under non-selective conditions, we followed the fate of a novel antibiotic resistance allele introduced into an ancestral donor population. While alleles in heteroplasmic donors segregated over time, conjugation produced almost only homoplasmic recipients whose allele composition closely mirrored that of the donor pool. Heteroplasmic recipients were rare and arose primarily from multiple plasmid transfer events prior to plasmid establishment. A mathematical model calibrated to the experimental setup predicts that conjugation accelerates plasmid allele segregation, with effects scaling with plasmid copy number and conjugation frequency. Our findings identify horizontal transfer as a previously unrecognized segregation pathway shaping the evolution of mobile genetic elements.

evolutionary biology↗

bioRxiv2025

SegMantX: a novel tool for detecting DNA duplications uncovers prevalent duplications in plasmids

Segmental duplications play an important role in genome evolution via their contribution to copy-number variation, gene-family diversification and the emergence of novel functions. The detection of segmental duplications is challenging due to heterogeneous amelioration of sequence similarity among duplicates, which hinders the reconstruction of continuous sequence alignment. Here we introduce SegMantX, a novel approach for the identifcation of diverged segmental duplications using local alignment chaining. In this approach, local alignments resulting from a preliminary sequence similarity search (e.g., BLASTn) are chained into continuous segments. Evaluating the performance of SegMantX simulated sequences shows that the tool can detect diverged duplications beyond the sensitivity limits of standard alignment-based methods. Applying SegMantX to 6,784 enterobacterial plasmids, we find that 74% plasmids contain duplicated regions, most of which correspond to duplicated mobile genetic elements (MGEs; e.g., transposons and insertion sequences). Furthermore, we demonstrate the applicability of SegMantX for the identification of diverged gene transfers between replicons, and plasmid hybridization events. Our findings highlight MGEs as drivers of segmental duplications in plasmid evolution, leading to the amplification of their cargo genes, including antibiotic resistance genes. SegMantX provides a powerful framework for reconstructing diverged segmental duplications and other alignment problems.

bioinformatics↗

bioRxiv2025

Persistence of large conjugative plasmids relies on the combination of active partitioning and addiction mechanisms

Extrachromosomal and mobile genetic elements, including transposable elements, plasmids, and accessory chromosomes, are found across all domains of life. The replication and segregation of elements integrated into the host chromosome are ensured by the host life cycle. In contrast, the persistence of autonomously replicating elements relies on their ability to remain within the host population. Here, we compare the evolutionary advantage of different persistence strategies found in prokaryotic plasmids. Through intracellular competitions between plasmid genotypes, we find that the combination of active partitioning during cell division with a toxin-antitoxin (TA) system for post-segregational killing increases plasmid fitness more than either strategy alone. Mathematical modeling of long-term plasmid evolution, calibrated with empirical plasmid loss dynamics, further supports these findings. A survey of enterobacterial genomes indicates that partitioning and TA systems are core features of large conjugative plasmids. Indeed, we confirm the presence of a previously unrecognized type I TA system in IncX3 plasmids, which serve as important vectors of antibiotic resistance in human pathogens. These findings suggest that large conjugative plasmids encode TA systems, including those yet to be identified. The combination of TA and partitioning systems emerges as the most effective strategy for the evolutionary success of low-copy extrachromosomal elements.

microbiology↗

bioRxiv2024

Evolution of plasmid domestication in plant-associated Pantoea: massive gain of genetic redundancy followed by differential gene loss

Plasmids are important drivers of evolutionary transformations and ecological adaptation in prokaryotes. Plasmids supplying the host with beneficial functions may become domesticated and gain a stable inheritance within the host lineage in the form of secondary chromosomes. Chromosomes descendent from plasmids (termed chromids) comprise core genes that are universally present in all taxon members. The origin of plasmid core genes remains poorly understood and alternative hypotheses entailing gene translocation or genetic redundancy are debated. Studying plasmid evolution in plant-associated Pantoea, we show that the large Pantoea plasmid (LPP) and plasmid pPag1 comprise core genes in the host taxa. We infer the LPP acquisition in plant-associated ancestors, while the pPag1 acquisition is traced to the ancestor of plant growth-promoting species. We show that both plasmids are vertically inherited and the LPP replication is furthermore coordinated with cell division, hence they are better described as chromids. Using phylogenomics we infer that the plasmid acquisition manifested in massive gene gain, some redundant with the chromosome, followed by differential gene loss. Our results provide an explanation for the evolution of core plasmid genes that entails plasmid-driven species divergence, which preliminarily manifests in genetic redundancy, and is followed by gradual loss of redundant genes. Significance statementAdaptation to host-associated lifestyle in bacteria can be promoted by the acquisition of plasmids, which are mobile extra-chromosomal genetic elements. A canonic example is the symbiosis Rhizobia plasmids, where essential traits to interact with Legumes are plasmid-encoded. Symbiosis plasmids are likely to evolve into stably inherited domesticated plasmids (termed chromids). Recent research reveals that potential chromids are widely distributed among plant-associated bacteria. Studying the origin of plasmids in the plant-associated genus Pantoea, we show that the large Pantoea plasmid (LPP) and plasmid pPag1 are domesticated. Phylogenomic analyses trace back the plasmid origin to ancestral nodes in the Pantoea species tree. We reconstruct main events in chromid evolution, including massive gene gain, some redundant with the chromosome, and differential gene loss.

evolutionary biology↗

bioRxiv2024

The evolution of antibiotic resistance islands occurs within the framework of plasmid lineages

Bacterial pathogens carrying multidrug resistance (MDR) plasmids are a major threat to human health. The acquisition of antibiotic resistance genes (ARGs) in plasmids is often facilitated by mobile genetic elements that copy or translocate ARGs between DNA molecules. The agglomeration of mobile elements in plasmids generates resistance islands comprising multiple ARGs. However, whether the emergence of resistance islands is restricted to specific MDR plasmid lineages remains understudied. Here we show that the agglomeration of ARGs in resistance islands is biased towards specific large plasmid lineages. Analyzing 6,784 plasmids in 2,441 Escherichia, Salmonella, and Klebsiella isolates, we quantify that 84% of the ARGs in MDR plasmids are found in resistance islands. We furthermore observe rapid evolution of ARG combinations in resistance islands. Most regions identified as resistance islands are shared among closely related plasmids but rarely among distantly related plasmids. Our results suggest the presence of barriers for the dissemination of ARGs between plasmid lineages, which are related to plasmid genetic properties, host range and the plasmid evolutionary history. The agglomeration of ARGs in plasmids is attributed to the workings of mobile genetic elements that operate within the framework of existing plasmid lineages.

evolutionary biology↗

bioRxiv2023

Heteroplasmy is rare in plant mitochondria compared to plastids despite similar mutation rates

Plant cells harbor two membrane-bound organelles containing their own genetic material - plastids and mitochondria. Although the two organelles co-exist and co-evolve within the same plant cells, they differ in genome copy number, intracellular organization, and mode of inheritance. How these attributes determine the time to fixation, or conversely, loss of neutral alleles is currently unresolved. Here we show that mitochondria and plastids share the same mutation rate yet plastid alleles remain in a heteroplasmic state significantly longer compared to mitochondrial alleles. By analysing genetic variants across populations of the marine flowering plant Zostera marina and simulating organelle allele dynamics, we examine the determinants of allele segregation and fixation time. Our results suggest that bottleneck on the cell population, e.g., during branching and seeding, and stratification of the meristematic tissue, are important determinants of mitochondrial allele dynamics. Furthermore, we suggest that the prolonged plastid allele dynamics are due to a yet unknown active plastid partition mechanism. The dissimilarity between plastid and mitochondrial novel allele fixation at different levels of organization may figure into differences in adaptation processes. Our study uncovers fundamental principles of organelle population genetics that are essential for further investigations of long-term evolution and molecular dating of divergence events.

bioinformatics↗

bioRxiv2023

Pseudogenes in plasmid genomes reveal past transitions in plasmid mobility

Evidence for gene non-functionalization due to mutational processes is found in genomes in the form of pseudogenes. Pseudogenes are known to be rare in prokaryote chromosomes, with the exception of lineages that underwent an extreme genome reduction (e.g., obligatory symbionts). Much less is known about the frequency of pseudogenes in prokaryotic plasmids; those are genetic elements that can transfer between cells and may encode beneficial traits for their host. Non-functionalization of plasmid-encoded genes may alter the plasmid characteristics, e.g., mobility, or their effect on the host. Analyzing 10, 832 prokaryotic genomes, we find that plasmid genomes are characterized by threefold-higher pseudogene density compared to chromosomes. The majority of plasmid pseudogenes correspond to deteriorated transposable elements. A detailed analysis of enterobacterial plasmids furthermore reveals frequent gene non-functionalization events associated with the loss of plasmid self-transmissibility. Reconstructing the evolution of closely related plasmids reveals that non-functionalization of the conjugation machinery led to the emergence of non-mobilizable plasmid types. Examples are virulence plasmids in Escherichia and Salmonella. Our study highlights non-functionalization of core plasmid mobility functions as one route for the evolution of domesticated plasmids. Pseudogenes in plasmids supply insights into past transitions in plasmid mobility that are akin to transitions in bacterial lifestyle.

evolutionary biology↗

bioRxiv2023

Worldwide population genomics reveal long-term stability of the mitochondrial chromosome composition in a keystone marine plant

Mitochondrial genomes (mitogenomes) of flowering plants are comprised of multiple chromosomes. Their copy number and composition can be dynamic within and among individual plants due to uneven replication of the chromosomes and homologous recombination. Nonetheless, despite their functional importance, the level of mitogenome conservation within species remains understudied. Whether the ontogenetic variation translates to evolution of mitogenome composition over generations is currently unknown. Here we show that the mitochondrial chromosome composition of the seagrass Zostera marina is conserved among worldwide populations that diverged ca 350,000 years ago. Using long-read sequencing we characterized the Z. marina mitochondrial genome and inferred the repertoire of recombination-induced configurations of its eight chromosomes. To characterize the chromosome composition worldwide and study its evolution we examined the mitogenome in Z. marina meristematic region sampled in 16 populations from the Pacific and Atlantic oceans. Our results reveal a striking similarity in the chromosome copy number suggesting stable equal proportions among distantly related populations and a high conservation of the mitochondrial genome within the plant germline, despite a notable variability during individual ontogenesis. Our study supplies a link between observations of dynamic mitogenomes at the level of plant individuals and long-term mitochondrial evolution. Significance statementExtensive studies on evolution of plant mitochondria in individual plants revealed great variability of the mitogenome architecture across tissues, however, data on the mitochondrion evolution at the population level is still scarce. We show that the mitochondrial genome architecture in a keystone marine plant, Zostera marina, remained conserved over ca. 350,000 years worldwide. We suggest that the extreme conservation of the Z. marina mitochondria is a manifestation of streamlined mitochondria inheritance over plant generations, e.g., via a plant germline.

genomics↗

bioRxiv2023

T6SS-mediated competitive exclusion among Pantoea agglomerans associated with plants

Establishment of symbiotic interactions with a host habitat depends on colonization success of the microbiome members. One route to success is increasing competitiveness against genotypes having similar adaptations. Many bacteria deploy the type VI secretion system (T6SS) to deliver toxic effector proteins into the cytoplasm of competing cells, whereas the attacking cells are protected from self-intoxication by immunity proteins. While the evolution of antagonistic interactions between species competing for similar niches is expected, the interactions between closely related strains having a similar core genetic makeup, remains understudied. Here we show that the strength of T6SS-mediated competition between closely related P. agglomerans strains is not associated with phylogenetic relatedness and rather depends on their effector and immunity genes repertoire. Annotating the T6SS components in eleven P. agglomerans isolates from wheat seeds, we observed a heterogeneous composition of effector and immunity determinants. Competition experiments further showed profound differences in the strain survival following reciprocal T6SS-mediated interactions. Reconstructing the evolution of T6SS loci across plant-associated P. agglomerans isolates indicates intra-chromosomal recombination as the main driver of effector and immunity gene diversification. Our results provide empirical data on intraspecific interactions during microbiota assembly likely to be at play during colonization of germinating plant seeds.

microbiology↗

bioRxiv2023

Closely related Bacteroides of the murine intestinal microbiota affect each other's growth positively or negatively

The mammalian intestine is a unique ecosystem for thousands of bacterial species and strains. How naturally coexisting bacteria of the microbiota interact with each other is not yet fully understood. Here, we isolated formerly coexisting, closely related strains of the genus Bacteroides from the intestines of healthy, wild-derived mice. The effect of one strain on another strains growth was tested in 169 pairs in vitro. We find a vast diversity of growth promoting and growth inhibiting activities. A strong positive effect was observed between two strains with differing metabolisms. Growth inhibition among a subset of strains was associated with the known bacterial toxin bacteroidetocin B. Across all strains, we observed growth promotion more often than growth inhibition. The effects were independent of two strains belonging to the same or different species. In some cases, one species differed in its effect on another according to host origin. These findings on obligate host-associated bacteria demonstrate that closely related and naturally coexisting strains have the potential to affect each others growth positively or negatively. These results have implications for our basic understanding of host-associated microbes and the design of synthetic microbial communities.

microbiology↗

bioRxiv2023

Segregational drift hinders the evolution of antibiotic resistance on polyploid replicons

The emergence of antibiotic resistance under treatment depends on the availability of resistance alleles and their establishment in the population. Novel resistance alleles are encoded either in chromosomal or extrachromosomal genetic elements; both types may be present in multiple copies within the cell. However, the effect of polyploidy on the emergence of antibiotic resistance remains understudied. Here we show that the establishment of resistance alleles in microbial populations depends on the ploidy level. Evolving bacterial populations under selection for antibiotic resistance, we demonstrate that resistance alleles in polyploid elements are lost frequently in comparison to alleles in monoploid elements due to segregational drift. Integrating the experiments with a mathematical model, we find an agreement between the theoretical and empirical allele dynamics, confirming our understanding of the allele segregation process. Using the mathematical model, we further show that the effect of polyploidy on the establishment probability of beneficial alleles is strongest for low replicon copy numbers and plateaus for high replicon copy numbers. Our results suggest that the distribution of fitness effects for mutations that get fixed in a population depends on the replicon ploidy level. Our study indicates that strategies for drug treatment of bacterial infections should take into consideration the pathogen ploidy level.

microbiology↗

bioRxiv2022

Ocean currents drive the worldwide colonization of the most widespread marine plant, eelgrass (Zostera marina)

Currents are unique drivers of oceanic phylogeography and so determine the distribution of marine coastal species, along with past glaciations and sea level changes. Here, we reconstruct the worldwide colonization history of eelgrass (Zostera marina L.), the most widely distributed marine flowering plant or seagrass from its origin in the Northwest Pacific, based on nuclear and chloroplast genomes. We identified two divergent Pacific clades with evidence for admixture along the East Pacific coast. Multiple west to east (trans-Pacific) colonization events support the key role of the North Pacific Current. Time-calibrated nuclear and chloroplast phylogenies yielded concordant estimates of the arrival of Z. marina in the Atlantic through the Canadian Arctic, suggesting that eelgrass-based ecosystems, hotspots of biodiversity and carbon sequestration, have only been present since [~]208 Kya (thousand years ago). Mediterranean populations were founded [~]53 Kya while extant distributions along western and eastern Atlantic shores coincide with the end of the Last Glacial Maximum ([~]20 Kya). The recent colonization and 5-to 7-fold lower genomic diversity of Atlantic compared to the Pacific populations raises concern and opportunity about how Atlantic eelgrass might respond to rapidly warming coastal oceans.

evolutionary biology↗

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

Denitrification in foraminifera has ancient origins and is complemented by associated bacteria

Benthic foraminifera are unicellular eukaryotes that inhabit sediments of aquatic environments. Several foraminifera of the order Rotaliida are known to store and use nitrate for denitrification, a unique energy metabolism among eukaryotes. The rotaliid Globobulimina spp. has been shown to encode an incomplete denitrification pathway of bacterial origin. However, the prevalence of denitrification genes in foraminifera remains unknown and the missing denitrification pathway components are elusive. Analysing transcriptomes and metagenomes of ten foraminiferal species from the Peruvian oxygen minimum zone, we show that denitrification genes are highly conserved in foraminifera. We infer of the last common ancestor of denitrifying foraminifera, which enables us to predict the ability to denitrify for additional foraminiferal species. Additionally, an examination of the foraminiferal microbiota reveals evidence for a stable interaction with Desulfobacteracea, which harbour genes that complement the foraminiferal denitrification pathway. Our results provide evidence that foraminiferal denitrification is complemented by the foraminifera-associated microbiome. The interaction of foraminifera with their resident bacteria is at the basis of foraminiferal adaptation to anaerobic environments that manifested in ecological success within oxygen depleted habitats. Significance StatementA substantial component of the global nitrogen cycle is the production of biologically inaccessible dinitrogen attributed to anaerobic denitrification by prokaryotes. Recent evidence identified a eukaryote - foraminifera - as new key players in this loss of bioavailable nitrogen. The evolution of denitrification in eukaryotes constitutes a rare event and the genetic mechanisms of the denitrification pathway in foraminifera are just starting to be elucidated. We present large-scale sequencing analyses of ten denitrifying foraminiferal species, which reveals the high conservation of the foraminiferal denitrification pathway. We further find evidence for a complementation of denitrification by the foraminiferal microbiome. Together, these findings provide novel insights on the early evolution of a previously overlooked component in the marine nitrogen cycle.

evolutionary biology↗

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

Natural competence in Chlorogloeopsis fritschii PCC 6912 and other ramified cyanobacteria

Lateral DNA transfer plays an important role in the evolution of genetic diversity in prokaryotes. DNA acquisition via transformation involves the uptake of DNA from the environment. The ability of recipient cells to actively transport DNA into the cytoplasm - termed natural competence - depends on the presence of type IV pili and competence proteins. Natural competence has been described in cyanobacteria for several organisms including unicellular and filamentous species. However, the presence of natural competence in ramified cyanobacteria, which are considered the peak of cyanobacterial morphological complexity, remains unknown. Here we show that ramified cyanobacteria harbour the genes essential for natural competence and experimentally demonstrate natural competence in the ramified cyanobacterium Chlorogloeopsis fritschii PCC 6912 (hereafter Chlorogloeopsis). Searching for homologs to known natural competence genes in ramified cyanobacteria showed that these genes are conserved in the majority of tested isolates. Experimental validation of natural competence using several alternative protocols demonstrates that Chlorogloeopsis could be naturally transformed with a replicative plasmid. Our results show that natural competence is a common trait in ramified cyanobacteria and that natural transformation is likely to play an important role in cyanobacteria evolution. ImportanceCyanobacteria are crucial players in the global biogeochemical cycles where they contribute to CO2- and N2-fixation. Their main ecological significance is the oxygen-producing photosynthetic apparatus that contributes to contemporary food chains. Ramified cyanobacteria form true-branching and multiseriate cell filament structures that represent a peak of prokaryotic multicellularity. Species in that group inhabit fresh and marine water habitats, thermal springs, arid environments, as well as endolithic and epiphytic habitats. Here we show that ramified cyanobacteria harbor the mechanisms required for DNA acquisition via natural transformation. The prevalence of mechanisms for natural uptake of DNA has implications for the role of DNA acquisition in the evolution of cyanobacteria. Furthermore, presence of mechanisms for natural transformation in ramified cyanobacteria opens up new possibilities for genetic modification of ramified cyanobacteria.

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↗