A High-Resolution View of Adaptive Event Dynamics in a Plasmid

Abstract

Coadaptation between bacterial hosts and plasmids frequently results in adaptive changes restricted exclusively to host genome leaving plasmids unchanged. To better understand this remarkable stability, we transformed naïve Escherichia coli cells with a plasmid carrying an antibiotic-resistance gene and forced them to adapt in a turbidostat environment. We then drew population samples at regular intervals and subjected them to duplex sequencing—a technique specifically designed for identification of low-frequency mutations. Variants at ten sites implicated in plasmid copy number control emerged almost immediately, tracked consistently across the experiment’s time points, and faded below detectable frequencies toward the end. This variation crash coincided with the emergence of mutations on the host chromosome. Mathematical modeling of trajectories for adaptive changes affecting plasmid copy number showed that such mutations cannot readily fix or even reach appreciable frequencies. We conclude that there is a strong selection against alterations of copy number even if it can provide a degree of growth advantage. This incentive is likely rooted in the complex interplay between mutated and wild-type plasmids constrained within a single cell and underscores the importance of understanding of intracellular plasmid variability.

Lineage tracing via associative chromosome/plasmid barcoding with siBar

“…We are planning to apply siBar to compare the effects of taking these two selection strategies. In our previous work, we have observed two types of variants on the plasmid pBR322, both of which boosted the antibiotics resistance levels of their host cells by increasing the plasmid copy number, thus achieving higher expression levels of the antibiotics resistance gene (Mei et al 2019). The difference between these two types of variants is that one type (variants at positions 3,027–3,035) only affected the copy number of the mutant plasmids carrying the variants, thus conferring mutant plasmids differential fitness.…”

“…To answer this question it is necessary to identify emerging mutations and trace them through time. Previously, we have applied duplex sequencing to identify emerging adaptive changes in a plasmid conferring antibiotic resistance to Escherichia coli cells maintained in a continuous turbidostat culture (Mei et al 2019). This work showed the emergence of nucleotide changes affecting the copy number control mechanism.…”

Freely accessible ready to use global infrastructure for SARS-CoV-2 monitoring

“…In this dataset a population of E. coli cells transformed with pBR322 is maintained in a turbidostat culture for an extensive period of time. Adaptive changes accumulated within the plasmid are then revealed with duplex sequencing 30 . Duplex sequencing allows identification of AVs at very low frequencies.…”

“…For the following analysis we selected two data points from Ref. 30 : one corresponding to the beginning of the experiment (s0) and the other to the end (s5). The first sample is expected to be nearly clonal with no variation, while the latter contains a number of adaptive changes with frequencies around 1%.…”

Mathematical Models of Plasmid Population Dynamics

“…However, large numbers of plasmids can also be detrimental as they produce a significant reduction in the growth rate of their bacterial hosts. This trade-off between fitness cost and plasmid stability has been shown, both in vitro and in theory, to result in the existence of an optimal plasmid copy-number susceptible to be tuned by evolution, with strong selection against increasing the number of plasmids carried in each cell ( Mei et al, 2019 ). A modeling study showed that selection against extreme copy-number plasmids can produce ecological cycles between mutants with low, intermediate, and high copy numbers, an eco-evolutionary process that can theoretically increase plasmid stability ( Watve et al, 2010 ).…”