This seemingly simple question — whether plasmids choose to go big or go multiple — ignited this project during a conversation about plasmids with Jero, my thesis supervisor. What began as a curiosity quickly evolved into a deep investigation, as we soon realized that the answer could unlock many new mysteries about plasmid biology.
Plasmids are circular DNA molecules that live independently of the bacterial chromosome, providing their hosts with powerful advantages, such as antibiotic resistance genes, virulence factors, and the ability to metabolize unusual substances. One fascinating feature of plasmids is that they can be present in multiple copies within a single cell, thereby amplifying their genetic content and evolutionary potential. This property has fascinated me ever since I began my PhD four years ago at the Evodynamics Lab.
Filling the Gaps
Early on, we identified a major gap in plasmid biology: although most plasmids exist in multiple copies, surprisingly little is known about their actual copy number in natural contexts; much of what we do know about plasmid copy number (PCN) comes from studies of well-characterized synthetic plasmids, while natural plasmids, the ones thriving in real-world bacterial communities, remained largely unexplored. To fill this gap, we undertook one of the first large-scale global analyses of PCNs, using public databases to analyze over 6,300 plasmids from clinically relevant bacteria, such as all members of the ESKAPEE group (Enterococcus, Staphylococcus, Klebsiella, Acinetobacter, Pseudomonas, Enterobacter and Escherichia). The acronym “ESKAPEE” highlights their ability to "escape" the effects of standard antibiotics, making them particularly dangerous in clinical settings. Therefore, they are perfect candidates for studying plasmid-mediated resistance and adaptation.
We found that plasmids of these bacteria exhibit remarkable diversity, not only in their structure but also in their copy numbers, which vary significantly across different species and lifestyles. Our analysis revealed a clear bimodal pattern in the distribution of plasmid copy numbers, reflecting two distinct strategies: high-copy plasmids and low-copy plasmids. High-copy plasmids have flexible and relaxed replication control, allowing them to serve as adaptable platforms. In contrast, low-copy plasmids maintain tight control over replication to ensure stable gene expression.
A pervasive scaling law rules plasmid biology
When we examined the factors that determine PCN, one notable pattern emerged: plasmid size proved to be the most significant predictor. Through the use of machine learning models, we found that copy number and plasmid size are connected by a universal scaling law across different genera. Specifically, as plasmids increase in size, their copy number decreases. This indicates that, despite the vast diversity of plasmids, they all adhere to a straightforward principle that balances size and abundance.
Additionally, this scaling results in a remarkably consistent proportion of plasmid DNA relative to the host chromosome, with each plasmid typically constituting about 2.5% of the total DNA of the chromosome. Interestingly, the overall plasmid DNA content in any given cell scales linearly with the number of plasmids it contains. This discovery provides insight into the metabolic balance that bacteria maintain with their plasmids and offers a simple method for estimating plasmid copy number based on size, which is crucial for understanding plasmid biology in natural and clinical contexts.
Fostering collaborative science
One of the most inspiring moments of this journey was presenting our work at the International Symposium on Plasmid Biology in Hamamatsu (Japan), a city renowned for its rich musical instrument heritage, precision technology industries, and its beautiful natural surroundings.
There, we met Lingchong You, who independently uncovered the same intriguing inverse relationship between plasmid size and copy number. This meeting sparked an incredible synergy between our groups, leading us to submit our complementary papers back-to-back. Our collaboration beautifully reflects a core value of Japanese culture known as wa (和), or harmony, where different parts come together to form a balanced whole. It's a nice reminder that science, at its best, is a team effort built on respect, trust, and a common goal.
Closing the circle
From that first question — should plasmids go big or go multiple? — we discovered that plasmids indeed face this evolutionary choice. Yet their strategies, whether high-copy or low-copy, converge on an elegant rule that balances size and abundance. Their strategies reflect a fundamental principle of evolution: balance. By adjusting size and copy number, plasmids manage the trade-offs between stability and host burden, reminding us that in the microbial world there’s no single path to success, but rather diverse solutions to the challenge of survival.