The extent and characteristics of DNA transfer between plasmids and chromosomes.

Plasmids are extrachromosomal genetic elements that reside in prokaryotes. The acquisition of plasmids encoding beneficial traits can facilitate short-term survival in harsh environmental conditions or long-term adaptation of new ecological niches. Due to their ability to transfer between cells, plasmids are considered agents of gene transfer. Nonetheless, the frequency of DNA transfer between plasmids and chromosomes remains understudied. Using a novel approach for detection of homologous loci between genome pairs, we uncover gene sharing with the chromosome in 1,974 (66%) plasmids residing in 1,016 (78%) taxonomically diverse isolates. The majority of homologous loci correspond to mobile elements, which may be duplicated in the host chromosomes in tens of copies. Neighboring shared genes often encode similar functional categories, indicating the transfer of multigene functional units. Rare transfer events of antibiotics resistance genes are observed mainly with mobile elements. The frequent erosion of sequence similarity in homologous regions indicates that the transferred DNA is often devoid of function. DNA transfer between plasmids and chromosomes thus generates genetic variation that is akin to workings of endosymbiotic gene transfer in eukaryotic evolution. Our findings imply that plasmid contribution to gene transfer most often corresponds to transfer of the plasmid entity rather than transfer of protein-coding genes between plasmids and chromosomes.

Heteroplasmy Is Rare in Plant Mitochondria Compared with Plastids despite Similar Mutation Rates.

Plant cells harbor two membrane-bound organelles containing their own genetic material-plastids and mitochondria. Although the two organelles coexist and coevolve within the same plant cells, they differ in genome copy number, intracellular organization, and mode of segregation. How these attributes affect 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 with mitochondrial alleles. By analyzing genetic variants across populations of the marine flowering plant Zostera marina and simulating organelle allele dynamics, we examine the determinants of allele segregation and allele fixation. Our results suggest that the bottlenecks on the cell population, e.g. during branching or 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 manifest in 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.

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.

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 6784 plasmids in 2441 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.

Assessing Functional Cognition and Health During COVID-19 Pandemic: Gender Differences Among Community-Dwelling Adults.

INTRODUCTION/OBJECTIVES: The COVID-19 pandemic has long-term implications for adult health and function, whether or not people were infected with the disease. Although cognitive disruptions are among the major symptoms of COVID-19, most research focused on managing medical symptoms, such as respiratory symptoms or pain. Thus, less is known about the pandemic's long-term implications for assessing functional cognition. This study aimed to examine COVID-19's effects on community-dwelling adults' functional cognition and health, comparing gender differences. METHODS: This cross-sectional study divided 118 community-dwelling adults (25 previously infected with COVID-19) into gender groups. Primary outcome measures included the Daily Living Questionnaire (DLQ) and short form health status survey, SF-12. RESULTS: No significant differences were found in functional cognition or health between participants who had contracted COVID-19 and those who remained healthy, but men had better functional cognition and health measures in comparison with women. CONCLUSIONS: Gender differences in functional cognition and health state may relate to gender-based family roles. It is essential to assess functional cognition of young adults who were exposed to a pandemic, such as COVID-19, because it may significantly affect their health and functional status. The DLQ is a reliable, valid assessment of functional cognition that may suit individuals who previously contracted COVID-19.

Role of natural transformation in the evolution of small cryptic plasmids in Synechocystis sp. PCC 6803.

Small cryptic plasmids have no clear effect on the host fitness and their functional repertoire remains obscure. The naturally competent cyanobacterium Synechocystis sp. PCC 6803 harbours several small cryptic plasmids; whether their evolution with this species is supported by horizontal transfer remains understudied. Here, we show that the small cryptic plasmid DNA is transferred in the population exclusively by natural transformation, where the transfer frequency of plasmid-encoded genes is similar to that of chromosome-encoded genes. Establishing a system to follow gene transfer, we compared the transfer frequency of genes encoded in cryptic plasmids pCA2.4 (2378 bp) and pCB2.4 (2345 bp) within and between populations of two Synechocystis sp. PCC 6803 labtypes (termed Kiel and Sevilla). Our results reveal that plasmid gene transfer frequency depends on the recipient labtype. Furthermore, gene transfer via whole plasmid uptake in the Sevilla labtype ranged among the lowest detected transfer rates in our experiments. Our study indicates that horizontal DNA transfer via natural transformation is frequent in the evolution of small cryptic plasmids that reside in naturally competent organisms. Furthermore, we suggest that the contribution of natural transformation to cryptic plasmid persistence in Synechocystis is limited.

Worldwide Population Genomics Reveal Long-Term Stability of the Mitochondrial Genome Architecture in a Keystone Marine Plant.

Mitochondrial genomes (mitogenomes) of flowering plants are composed of multiple chromosomes. Recombination within and between the mitochondrial chromosomes may generate diverse DNA molecules termed isoforms. The isoform copy number and composition can be dynamic within and among individual plants due to uneven replication 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 mitogenome 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. To characterize the mitochondrial genome architecture 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 isoform relative copy number, indicating a high conservation of the mitogenome composition among distantly related populations and 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.

SepT, a novel protein specific to multicellular cyanobacteria, influences peptidoglycan growth and septal nanopore formation in Anabaena sp. PCC 7120.

IMPORTANCE: Multicellular organization is a requirement for the development of complex organisms, and filamentous cyanobacteria such as Anabaena represent a paradigmatic case of bacterial multicellularity. The Anabaena filament can include hundreds of communicated cells that exchange nutrients and regulators and, depending on environmental conditions, can include different cell types specialized in distinct biological functions. Hence, the specific features of the Anabaena filament and how they are propagated during cell division represent outstanding biological issues. Here, we studied SepT, a novel coiled-coil-rich protein of Anabaena that is located in the intercellular septa and influences the formation of the septal specialized structures that allow communication between neighboring cells along the filament, a fundamental trait for the performance of Anabaena as a multicellular organism.

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 a remarkable agreement between the theoretical and empirical results, 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 are eventually fixed in a population depends on the replicon ploidy level. Our study indicates that the emergence of antibiotic resistance in bacterial pathogens depends on the pathogen ploidy level.

Ocean current patterns drive the worldwide colonization of eelgrass (Zostera marina).

Currents are unique drivers of oceanic phylogeography and thus 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. Two 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 there for ~243 ky (thousand years). Mediterranean populations were founded ~44 kya, while extant distributions along western and eastern Atlantic shores were founded at the end of the Last Glacial Maximum (~19 kya), with at least one major refuge being the North Carolina region. The recent colonization and five- to sevenfold lower genomic diversity of the Atlantic compared to the Pacific populations raises concern and opportunity about how Atlantic eelgrass might respond to rapidly warming coastal oceans.

Phylogenomic Testing of Root Hypotheses.

The determination of the last common ancestor (LCA) of a group of species plays a vital role in evolutionary theory. Traditionally, an LCA is inferred by the rooting of a fully resolved species tree. From a theoretical perspective, however, inference of the LCA amounts to the reconstruction of just one branch-the root branch-of the true species tree and should therefore be a much easier task than the full resolution of the species tree. Discarding the reliance on a hypothesized species tree and its rooting leads us to reevaluate what phylogenetic signal is directly relevant to LCA inference and to recast the task as that of sampling the total evidence from all gene families at the genomic scope. Here, we reformulate LCA and root inference in the framework of statistical hypothesis testing and outline an analytical procedure to formally test competing a priori LCA hypotheses and to infer confidence sets for the earliest speciation events in the history of a group of species. Applying our methods to two demonstrative data sets, we show that our inference of the opisthokonta LCA is well in agreement with the common knowledge. Inference of the proteobacteria LCA shows that it is most closely related to modern Epsilonproteobacteria, raising the possibility that it may have been characterized by a chemolithoautotrophic and anaerobic life style. Our inference is based on data comprising between 43% (opisthokonta) and 86% (proteobacteria) of all gene families. Approaching LCA inference within a statistical framework renders the phylogenomic inference powerful and robust.

Out of the 'host' box: extreme off-host conditions alter the infectivity and virulence of a parasitic bacterium.

Disease agents play an important role in the ecology and life history of wild and cultivated populations and communities. While most studies focus on the adaptation of parasites to their hosts, the adaptation of free-living parasite stages to their external (off-host) environment may tell us a lot about the factors that shape the distribution of parasites. Pasteuria ramosa is an endoparasitic bacterium of the water flea Daphnia with a wide geographical distribution. Its transmission stages rest outside of the host and thus experience varying environmental regimes. We examined the life history of P. ramosa populations from four environmental conditions (i.e. groups of habitats): the factorial combinations of summer-dry water bodies or not, and winter-freeze water bodies or not. Our goal was to examine how the combination of winter temperature and summer dryness affects the parasite's ability to attach to its host and to infect it. We subjected samples of the four groups of habitats to temperatures of 20, 33, 46 and 60°C in dry and wet conditions, and exposed a susceptible clone of Daphnia magna to the treated spores. We found that spores which had undergone desiccation endured higher temperatures better than spores kept wet, both regarding attachment and subsequent infection. Furthermore, spores treated with heightened temperatures were much less infective and virulent. Even under high temperatures (60°C), exposed spores from all populations were able to attach to the host cuticle, albeit they were unable to establish infection. Our work highlights the sensitivity of a host-free resting stage of a bacterial parasite to the external environment. Long heatwaves and harsh summers, which are becoming more frequent owing to recent climate changes, may therefore pose a problem for parasite survival. This article is part of the theme issue 'Infectious disease ecology and evolution in a changing world'.

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 2 levels: the phenotype and the genotype. Our results show that depending on the mode of replication and segregation, the fixation of the mutant phenotype may precede genotypic fixation 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. Within the heterozygosity window, the population is phenotypically adapted, while both alleles persist in the population. Replicon ploidy thus allows for the maintenance of genetic variation following phenotypic adaptation and consequently for reversibility in adaptation to fluctuating environmental conditions.

Natural Competence in the Filamentous, Heterocystous Cyanobacterium Chlorogloeopsis fritschii PCC 6912.

Lateral gene transfer plays an important role in the evolution of genetic diversity in prokaryotes. DNA transfer via natural transformation depends on the ability of recipient cells to actively transport DNA from the environment into the cytoplasm, termed natural competence, which relies on the presence of type IV pili and other competence proteins. Natural competence has been described in cyanobacteria for several organisms, including unicellular and filamentous species. However, natural competence in cyanobacteria that differentiate specialized cells for N2-fixation (heterocysts) and form branching or multiseriate cell filaments (termed subsection V) remains unknown. Here, we show that genes essential for natural competence are conserved in subsection V cyanobacteria. Furthermore, using the replicating plasmid pRL25C, we experimentally demonstrate natural competence in a subsection V organism: Chlorogloeopsis fritschii PCC 6912. Our results suggest that natural competence is a common trait in cyanobacteria forming complex cell filament morphologies. IMPORTANCE Cyanobacteria are crucial players in the global biogeochemical cycles, where they contribute to CO2- and N2-fixation. Their main ecological significance is the primary biomass production owing to oxygenic photosynthesis. Cyanobacteria are a diverse phylum, in which the most complex species differentiate specialized cell types and form true-branching or multiseriate cell filament structures (termed subsection V cyanobacteria). These bacteria are considered a peak in the evolution of prokaryotic multicellularity. Among others, species in that group inhabit fresh and marine water habitats, soil, and extreme habitats such as thermal springs. Here, we show that the core genes required for natural competence are frequent in subsection V cyanobacteria and demonstrate for the first time natural transformation in a member of subsection V. The prevalence of natural competence has implications for the role of DNA acquisition in the genome evolution of cyanobacteria. Furthermore, the presence of mechanisms for natural transformation opens up new possibilities for the genetic modification of subsection V cyanobacteria.

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 symbiont 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 with core genes, accessory genes are enriched in genome plasticity functions. We found no evidence for recent HGT 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 in comparison to that of SOX. Our results show that the pangenomes of both symbionts evolved mainly by vertical inheritance. We conclude that genome evolution of environmentally transmitted symbionts that associate with individual hosts over their lifetime is affected by a narrow symbiosis where the frequency of HGT is constrained.

Denitrification in foraminifera has an ancient origin 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. Analyzing transcriptomes and metagenomes of 10 foraminiferal species from the Peruvian oxygen minimum zone, we show that denitrification genes are highly conserved in foraminifera. We infer 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 Desulfobacteraceae, which harbor 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 in oxygen depleted habitats.

Gene sharing among plasmids and chromosomes reveals barriers for antibiotic resistance gene transfer.

The emergence of antibiotic resistant bacteria is a major threat to modern medicine. Rapid adaptation to antibiotics is often mediated by the acquisition of plasmids carrying antibiotic resistance (ABR) genes. Nonetheless, the determinants of plasmid-mediated ABR gene transfer remain debated. Here, we show that the propensity of ABR gene transfer via plasmids is higher for accessory chromosomal ABR genes in comparison with core chromosomal ABR genes, regardless of the resistance mechanism. Analysing the pattern of ABR gene occurrence in the genomes of 2635 Enterobacteriaceae isolates, we find that 33% of the 416 ABR genes are shared between chromosomes and plasmids. Phylogenetic reconstruction of ABR genes occurring on both plasmids and chromosomes supports their evolution by lateral gene transfer. Furthermore, accessory ABR genes (encoded in less than 10% of the chromosomes) occur more abundantly in plasmids in comparison with core ABR genes (encoded in greater than or equal to 90% of the chromosomes). The pattern of ABR gene occurrence in plasmids and chromosomes is similar to that in the total Escherichia genome. Our results thus indicate that the previously recognized barriers for gene acquisition by lateral gene transfer apply also to ABR genes. We propose that the functional complexity of the underlying ABR mechanism is an important determinant of ABR gene transferability. This article is part of the theme issue 'The secret lives of microbial mobile genetic elements'.

Segregational Drift Constrains the Evolutionary Rate of Prokaryotic Plasmids.

Plasmids are extrachromosomal genetic elements in prokaryotes that have been recognized as important drivers of microbial ecology and evolution. Plasmids are found in multiple copies inside their host cell where independent emergence of mutations may lead to intracellular genetic heterogeneity. The intracellular plasmid diversity is thus subject to changes upon cell division. However, the effect of plasmid segregation on plasmid evolution remains understudied. Here, we show that genetic drift during cell division-segregational drift-leads to the rapid extinction of novel plasmid alleles. We established a novel experimental approach to control plasmid allele frequency at the levels of a single cell and the whole population. Following the dynamics of plasmid alleles in an evolution experiment, we find that the mode of plasmid inheritance-random or clustered-is an important determinant of plasmid allele dynamics. Phylogenetic reconstruction of our model plasmid in clinical isolates furthermore reveals a slow evolutionary rate of plasmid-encoded genes in comparison to chromosomal genes. Our study provides empirical evidence that genetic drift in plasmid evolution occurs at multiple levels: the host cell and the population of hosts. Segregational drift has implications for the evolutionary rate heterogeneity of extrachromosomal genetic elements.