Molecular and Genome Evolution of Prokaryotic Plasmids

Project Information

pMolEvol

Grant agreement ID: 101043835

DOI

10.3030/101043835

EC signature date 31 October 2022

Start date 1 April 2023

End date 31 March 2028

Funded under

• European Research Council (ERC)

Total cost

€ 2 112 500,00

EU contribution

€ 2 112 500,00

Coordinated by

CHRISTIAN-ALBRECHTS-UNIVERSITAET ZU KIEL
Germany

Periodic Reporting for period 1 - pMolEvol (Molecular and Genome Evolution of Prokaryotic Plasmids)

Reporting period: 2023-04-01 to 2025-09-30

Summary of the context and overall objectives of the project

Plasmids are autonomously replicating extrachromosomal elements that play a key role in prokaryotic ecology and evolution. In the context of human health, they have been extensively studied for their role in spreading antibiotic resistance and virulence genes among pathogenic bacteria. To date, research on plasmid evolution has primarily focused on their biodiversity and their ecological and evolutionary impact on prokaryotic hosts. However, the fundamental processes underlying plasmid genome evolution remain largely unexplored. While much research has examined the diversity of plasmids and their effects on bacterial populations, the fundamental mechanisms shaping plasmid genome evolution are still poorly understood. Unlike free-living organisms, plasmids depend on their bacterial hosts for resources and reproduction. As a result, they are subject to evolutionary pressures on two levels: their ability to replicate and persist within individual host cells, and their impact on the fitness of those host cells within a population.
The pMolEvol project aims to build a new theoretical and experimental framework to understand how plasmids evolve at the molecular and genomic levels. Our key objectives are to: 1) to quantify the effect of multilevel drift and selection on plasmid genetic diversity, 2) supply a framework for quantification of plasmid fitness and its effect on plasmid evolution, 3) characterize patterns and rate of plasmid genome evolution. The pMolEvol research framework is grounded in empirical data from plasmid evolution experiments and phylogenomic reconstructions of past evolutionary events. The novel concepts and methodologies developed in pMolEvol offer new directions for expanding classical population genetics to include prokaryotic systems. Because the mechanisms of segregating, replicating, and translating genetic information are conserved across all domains of life, studying plasmids provides a powerful lens through which to explore general principles governing the evolution of autonomously replicating genetic elements. Ultimately, our goal is to trace plasmid evolution from emergence to extinction.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

The main achievements of the pMolEvol so far, structured under the three specific objectives:
1. DIVERSITY – Quantifying the effects of drift, selection, and transfer on plasmid diversity
• We developed and empirically validated a model of plasmid allele dynamics showing how copy number, replication, and segregation influence allele fixation, even for beneficial alleles.
• Demonstrated rapid loss of plasmid alleles due to segregational drift, even when mildly beneficial.
• Extended the plasmid model to mitochondria and plastids in Zostera marina, showing organellar allele longevity is shaped by copy number and segregation.
• Showed that lateral gene transfer by transformation affects both plasmid and chromosomal alleles equally in naturally competent cyanobacteria, modulated by genotype and environment.
2. FITNESS – Developing a framework to quantify plasmid fitness and evolution
• Designed intra-cellular competition experiments showing that plasmids with both partition (Par) and toxin-antitoxin (TA) systems are more stable and have higher fitness than those with only one system.
• Developed a predictive mathematical model for plasmid competition outcomes.
• Comparative genomics revealed Par and TA are core systems in enteric plasmids, and identified a previously unannotated TA system in the IncX group.
• Providing support for the idea that stability systems are essential for evolutionary success of low-copy extrachromosomal genetic elements.
3. TEMPO – Measuring rates and patterns of plasmid molecular evolution
• Found that antibiotic resistance genes (ARGs) cluster in resistance islands within specific plasmid lineages, suggesting limited horizontal ARG transfer due to lineage barriers and MGE dynamics.
• Characterized the evolution of plasmid domestication via loss of conjugation functions.
• Developed SegMantX, a tool for detecting segmental duplications in bacterial replicons.
• Showed that plasmid-chromosome gene transfer is rare, typically mediated by MGEs, and often involves non-functional DNA.
• Studied plasmids in Pantoea, showing vertical inheritance and synchronization of plasmid and chromosomal replication, consistent with domestication and plasmid-to-chromosome transition.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

We demonstrated the role of multi-level drift and selection in the evolution of extrachromosomal genetic elements, providing both empirical evidence and theoretical groundwork for new concepts in population genetics. Additionally, we identified key events in plasmid domestication, outlining potential pathways through which plasmids evolve into accessory chromosomes. Our work advances the population genetics of extrachromosomal elements beyond the current state of the art. Furthermore, by exploring plasmid domestication, we establish a foundation for identifying accessory chromosomes in bacterial taxa beyond the well-studied genera Vibrio and Rhizobia.