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.

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.

Colonization dynamics of Pantoea agglomerans in the wheat root habitat.

Plants are colonized by microbial communities that have diverse implications for plant development and health. The establishment of a stable plant-bacteria interaction depends on a continuous coexistence over generations. Transmission via the seed is considered as the main route for vertical inheritance of plant-associated bacteria. Nonetheless, the ecological principles that govern the plant colonization by seed endophytes remain understudied. Here we quantify the contribution of arrival time and colonization history to bacterial colonization of the wheat root. Establishing a common seed endophyte, Pantoea agglomerans, and wheat as a model system enabled us to document bacterial colonization of the plant roots during the early stages of germination. Using our system, we estimate the carrying capacity of the wheat roots as 108 cells g-1 , which is robust among individual plants and over time. Competitions in planta reveal a significant advantage of early incoming colonizers over late-incoming colonizers. Priming for the wheat environment had little effect on the colonizer success. Our experiments thus provide empirical data on the root colonization dynamics of a seed endophyte. The persistence of seed endophyte bacteria with the plant population over generations may contribute to the stable transmission that is one route for the evolution of a stable host-associated lifestyle.

Segregational Drift and the Interplay between Plasmid Copy Number and Evolvability.

The ubiquity of plasmids in all prokaryotic phyla and habitats and their ability to transfer between cells marks them as prominent constituents of prokaryotic genomes. Many plasmids are found in their host cell in multiple copies. This leads to an increased mutational supply of plasmid-encoded genes and genetically heterogeneous plasmid genomes. Nonetheless, the segregation of plasmid copies into daughter cells during cell division is considered to occur in the absence of selection on the plasmid alleles. We investigate the implications of random genetic drift of multicopy plasmids during cell division-termed here "segregational drift"-to plasmid evolution. Performing experimental evolution of low- and high-copy non-mobile plasmids in Escherichia coli, we find that the evolutionary rate of multicopy plasmids does not reflect the increased mutational supply expected according to their copy number. In addition, simulated evolution of multicopy plasmid alleles demonstrates that segregational drift leads to increased loss frequency and extended fixation time of plasmid mutations in comparison to haploid chromosomes. Furthermore, an examination of the experimentally evolved hosts reveals a significant impact of the plasmid type on the host chromosome evolution. Our study demonstrates that segregational drift of multicopy plasmids interferes with the retention and fixation of novel plasmid variants. Depending on the selection pressure on newly emerging variants, plasmid genomes may evolve slower than haploid chromosomes, regardless of their higher mutational supply. We suggest that plasmid copy number is an important determinant of plasmid evolvability due to the manifestation of segregational drift.

Antibiotics Interfere with the Evolution of Plasmid Stability.

Extra-chromosomal genetic elements are important drivers of bacterial evolution, and their evolutionary success depends on positive selection for the genes they encode. Examples are plasmids encoding antibiotic resistance genes that are maintained in the presence of antibiotics (e.g., [1-3]). Plasmid maintenance is considered a metabolic burden to the host [4]; hence, when the cost of plasmid carriage outweighs its benefit, plasmid-free segregants are expected to outcompete plasmid-carrying cells, eventually leading to plasmid loss [5-7]. Thus, in the absence of positive selection, plasmid survival hinges upon stable persistence in the population. The ubiquity of plasmids in nature suggests that plasmids having a negligible effect on host fitness may evolve stable inheritance and thus gain a long-term persistence in the population, also in the absence of positive selection [8]. Nonetheless, the transition of plasmids into stably inherited genetic elements remains understudied. Here, we show that positive selection for a plasmid-encoded gene interferes with the evolution of plasmid stability. Evolving plasmids under different selection regimes in Escherichia coli, we find that antibiotics led to plasmid amplification, resulting in plasmid instability. Thus, under positive selection, suboptimal solutions for plasmid stability were maintained in the population hindering long-term plasmid persistence. Indeed, a survey of Escherichia plasmids confirms that antibiotic resistance genes are rarely found on small plasmids. Our results show that a plasmid-mediated advantage for the host may manifest in reduced plasmid evolutionary success. Considering plasmids as autonomously evolving entities holds promise for understanding the factors that govern their evolution.

Intracellular Competitions Reveal Determinants of Plasmid Evolutionary Success.

Plasmids are autonomously replicating genetic elements that are ubiquitous in all taxa and habitats where they constitute an integral part of microbial genomes. The stable inheritance of plasmids depends on their segregation during cell division and their long-term persistence in a host population is thought to largely depend on their impact on the host fitness. Nonetheless, many plasmids found in nature are lacking a clear trait that is advantageous to their host; the determinants of plasmid evolutionary success in the absence of plasmid benefit to the host remain understudied. Here we show that stable plasmid inheritance is an important determinant of plasmid evolutionary success. Borrowing terminology from evolutionary biology of cellular living forms, we hypothesize that Darwinian fitness is key for the plasmid evolutionary success. Performing intracellular plasmid competitions between non-mobile plasmids enables us to compare the evolutionary success of plasmid genotypes within the host, i.e., the plasmid fitness. Intracellular head-to-head competitions between stable and unstable variants of the same model plasmid revealed that the stable plasmid variant has a higher fitness in comparison to the unstable plasmid. Preemptive plasmid competitions reveal that plasmid fitness may depend on the order of plasmid arrival in the host. Competitions between plasmids characterized by similar stability of inheritance reveal plasmid fitness differences depending on the plasmid-encoded trait. Our results further reveal that competing plasmids can be maintained in coexistence following plasmid fusions that maintain unstable plasmid variants over time. Plasmids are not only useful accessory genetic elements to their host but they are also evolving and replicating entities, similarly to cellular living forms. There is a clear link between plasmid genetics and plasmid evolutionary success - hence plasmids are evolving entities whose fitness is quantifiable.

Quantification of Plasmid-Mediated Antibiotic Resistance in an Experimental Evolution Approach.

Plasmids play a major role in microbial ecology and evolution as vehicles of lateral gene transfer and reservoirs of accessory gene functions in microbial populations. This is especially the case under rapidly changing environments such as fluctuating antibiotics exposure. We recently showed that plasmids maintain antibiotic resistance genes in Escherichia coli without positive selection for the plasmid presence. Here we describe an experimental system that allows following both the plasmid genotype and phenotype in long-term evolution experiments. We use molecular techniques to design a model plasmid that is subsequently introduced to an experimental evolution batch system approach in an E. coli host. We follow the plasmid frequency over time by applying replica plating of the E. coli populations while quantifying the antibiotic resistance persistence. In addition, we monitor the conformation of plasmids in host cells by analyzing the extent of plasmid multimer formation by plasmid nicking and agarose gel electrophoresis. Such an approach allows us to visualize not only the genome size of evolving plasmids but also their topological conformation-a factor highly important for plasmid inheritance. Our system combines molecular strategies with traditional microbiology approaches and provides a set-up to follow plasmids in bacterial populations over a long time. The presented approach can be applied to study a wide range of mobile genetic elements in the future.

Emergence of plasmid stability under non-selective conditions maintains antibiotic resistance.

Plasmid acquisition is an important mechanism of rapid adaptation and niche expansion in prokaryotes. Positive selection for plasmid-coded functions is a major driver of plasmid evolution, while plasmids that do not confer a selective advantage are considered costly and expected to go extinct. Yet, plasmids are ubiquitous in nature, and their persistence remains an evolutionary paradox. Here, we demonstrate that non-mobile plasmids persist over evolutionary timescales without selection for the plasmid function. Evolving a minimal plasmid encoding for antibiotics resistance in Escherichia coli, we discover that plasmid stability emerges in the absence of antibiotics and that plasmid loss is determined by transcription-replication conflicts. We further find that environmental conditions modulate these conflicts and plasmid persistence. Silencing the transcription of the resistance gene results in stable plasmids that become fixed in the population. Evolution of plasmid stability under non-selective conditions provides an evolutionary explanation for the ubiquity of plasmids in nature.

Carrying Capacity and Colonization Dynamics of Curvibacter in the Hydra Host Habitat.

Most eukaryotic species are colonized by a microbial community - the microbiota - that is acquired during early life stages and is critical to host development and health. Much research has focused on the microbiota biodiversity during the host life, however, empirical data on the basic ecological principles that govern microbiota assembly is lacking. Here we quantify the contribution of colonizer order, arrival time and colonization history to microbiota assembly on a host. We established the freshwater polyp Hydra vulgaris and its dominant colonizer Curvibacter as a model system that enables the visualization and quantification of colonizer population size at the single cell resolution, in vivo, in real time. We estimate the carrying capacity of a single Hydra polyp as 2 × 105Curvibacter cells, which is robust among individuals and time. Colonization experiments reveal a clear priority effect of first colonizers that depends on arrival time and colonization history. First arriving colonizers achieve a numerical advantage over secondary colonizers within a short time lag of 24 h. Furthermore, colonizers primed for the Hydra habitat achieve a numerical advantage in the absence of a time lag. These results follow the theoretical expectations for any bacterial habitat with a finite carrying capacity. Thus, Hydra colonization and succession processes are largely determined by the habitat occupancy over time and Curvibacter colonization history. Our experiments provide empirical data on the basic steps of host-associated microbiota establishment - the colonization stage. The presented approach supplies a framework for studying habitat characteristics and colonization dynamics within the host-microbe setting.