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 ...

Kiel University

Plasmids are extra-chromosomal genetic elements whose ecology and evolution depend on their genetic repertoire and interaction with the host. We review the events that lead to transitions between plasmid lifestyle modes - invasion, host range, plasmid persistence and adaptation - from a plasmid perspective. Plasmid lifestyle is determined by various traits, including mobility, stability and indispensability that vary in their magnitude. Transitions between the plasmid lifestyles, invasion, host range, plasmid persistence and adpatation, are caused by the interplay between plasmid traits and host biology. Mobility and indispensability are important in plasmid ecology, whereas plasmid stability is more relevant for long-term plasmid evolution. In transitioning into additional chromosomes plasmids loose their independence and enter the host lineage. Though plasmids are confined to their hosts, their evolution may be independent of prokaryotic chromosomes.

An evolutionary perspective on plasmid lifestyle modes.

Benthic foraminifera are unicellular eukaryotes inhabiting sediments of aquatic environments. Several species were shown to store and use nitrate for complete denitrification, a unique energy metabolism among eukaryotes. The population of benthic foraminifera reaches high densities in oxygen-depleted marine habitats, where they play a key role in the marine nitrogen cycle. However, the mechanisms of denitrification in foraminifera are still unknown, and the possibility of a contribution of associated bacteria is debated. Here, we present evidence for a novel eukaryotic denitrification pathway that is encoded in foraminiferal genomes. Large-scale genome and transcriptomes analyses reveal the presence of a denitrification pathway in foraminifera species of the genus Globobulimina. This includes the enzymes nitrite reductase (NirK) and nitric oxide reductase (Nor) as well as a wide range of nitrate transporters (Nrt). A phylogenetic reconstruction of the enzymes' evolutionary history uncovers evidence for an ancient acquisition of the foraminiferal denitrification pathway from prokaryotes. We propose a model for denitrification in foraminifera, where a common electron transport chain is used for anaerobic and aerobic respiration. The evolution of hybrid respiration in foraminifera likely contributed to their ecological success, which is well documented in palaeontological records since the Cambrian period.

A Novel Eukaryotic Denitrification Pathway in Foraminifera.

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.

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

Benthic foraminifera populate a diverse range of marine habitats. Their ability to use alternative electron acceptors-nitrate (NO) or oxygen (O)-makes them important mediators of benthic nitrogen cycling. Nevertheless, the metabolic scaling of the two alternative respiration pathways and the environmental determinants of foraminiferal denitrification rates are yet unknown. We measured denitrification and O respiration rates for 10 benthic foraminifer species sampled in the Peruvian oxygen minimum zone (OMZ). Denitrification and O respiration rates significantly scale sublinearly with the cell volume. The scaling is lower for O respiration than for denitrification, indicating that NO metabolism during denitrification is more efficient than O metabolism during aerobic respiration in foraminifera from the Peruvian OMZ. The negative correlation of the O respiration rate with the surface/volume ratio is steeper than for the denitrification rate. This is likely explained by the presence of an intracellular NO storage in denitrifying foraminifera. Furthermore, we observe an increasing mean cell volume of the Peruvian foraminifera, under higher NO availability. This suggests that the cell size of denitrifying foraminifera is not limited by O but rather by NO availability. Based on our findings, we develop a mathematical formulation of foraminiferal cell volume as a predictor of respiration and denitrification rates, which can further constrain foraminiferal biogeochemical cycling in biogeochemical models. Our findings show that NO is the preferred electron acceptor in foraminifera from the OMZ, where the foraminiferal contribution to denitrification is governed by the ratio between NO and O.

Metabolic preference of nitrate over oxygen as an electron acceptor in foraminifera from the Peruvian oxygen minimum zone.

In experimental evolution, scientists evolve organisms in the lab, typically by challenging them to new environmental conditions. How best to evolve a desired trait? Should the challenge be applied abruptly, gradually, periodically, sporadically? Should one apply chemical mutagenesis, and do strains with high innate mutation rate evolve faster? What are ideal population sizes of evolving populations? There are endless strategies, beyond those that can be exposed by individual labs. We therefore arranged a community challenge, Evolthon, in which students and scientists from different labs were asked to evolve Escherichia coli or Saccharomyces cerevisiae for an abiotic stress-low temperature. About 30 participants from around the world explored diverse environmental and genetic regimes of evolution. After a period of evolution in each lab, all strains of each species were competed with one another. In yeast, the most successful strategies were those that used mating, underscoring the importance of sex in evolution. In bacteria, the fittest strain used a strategy based on exploration of different mutation rates. Different strategies displayed variable levels of performance and stability across additional challenges and conditions. This study therefore uncovers principles of effective experimental evolutionary regimens and might prove useful also for biotechnological developments of new strains and for understanding natural strategies in evolutionary arms races between species. Evolthon constitutes a model for community-based scientific exploration that encourages creativity and cooperation.

Evolthon: A community endeavor to evolve lab evolution.

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.

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

Population bottlenecks leading to a drastic reduction of the population size are common in the evolutionary dynamics of natural populations; their occurrence is known to have implications for genome evolution due to genetic drift, the consequent reduction in genetic diversity, and the rate of adaptation. Nevertheless, an empirical characterization of the effect of population bottleneck size on evolutionary dynamics of bacteria is currently lacking. In this study, we show that selective conditions have a stronger effect on the evolutionary history of bacteria in comparison to population bottlenecks. We evolved Escherichia coli populations under three different population bottleneck sizes (small, medium, and large) in two temperature regimes (37 °C and 20 °C). We find a high genetic diversity in the large in comparison to the small bottleneck size. Nonetheless, the cold temperature led to reduced genetic diversity regardless the bottleneck size; hence, the temperature has a stronger effect on the genetic diversity in comparison to the bottleneck size. A comparison of the fitness gain among the evolved populations reveals a similar pattern where the temperature has a significant effect on the fitness. Our study demonstrates that population bottlenecks are an important determinant of bacterial evolvability; their consequences depend on the selective conditions and are best understood via their effect on the standing genetic variation.

Tanita Wein

Institute of Microbiology

Kiel University

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

Tanita Wein

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Institute of Microbiology

Kiel University

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

Institute of Microbiology