Segregational Drift Hinders the Evolution of Antibiotic Resistance on Polyploid Replicons
Ana Garoña, Mario Santer, Nils F. Hülter, Hildegard Uecker,Tal Dagan
Abstract
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.
Introduction
Bacterial adaptation to novel environmental conditions depends on the availability of beneficial alleles and the dynamics of their proliferation within the population. However, novel alleles are initially rare in the population and therefore–even if they are adaptive–prone to stochastic loss due to genetic drift [1]. The probability that beneficial alleles survive the early proliferation phase and become established is termed ’establishment probability’. The probability of establishment (and eventually fixation) of beneficial alleles is a key concept in theoretical population genetics [2,3], dating back to the very early days of the field [1]. Understanding the determinants of allele establishment in bacterial populations is pivotal in the context of antibiotic resistance evolution. Antibiotic treatment confers a growth advantage to bacteria carrying resistance alleles, which might establish and subsequently rise to high numbers. The emergence of novel resistant strains of diverse human and livestock pathogens due to antibiotic treatment has worrisome consequences for global human health [4]. Sustainable treatment strategies are needed [5], for the development of which a profound understanding of the dynamics of beneficial alleles–including the early stochastic phase–is necessary.
Materials and methods
Bacterial strains, plasmids and culture conditions
The A. baylyi strain BD413 (DSM No. 588, German Collection of Microorganisms and Cell Cultures, DSMZ) also known as strain ADP1 (GenBank accession no. NC_005966.1) was used as the model organism in all experiments. A. baylyi is a monoploid species with a single copy of its chromosome [41,42]. For the chromosomal allele experiments, A. baylyi strain BD4 was also used. Either A. baylyi BD413 or BD4 were used during plasmid or chromosomal modification constructions. Primers used in this study are listed in supplementary tables (S1 Table). A. baylyi was propagated at 30°C in LB medium in liquid shaking cultures or plates. For molecular cloning and plating, growth limiting factors, such as antibiotics, for the selection of plasmid/mutation carrying cells were used at the following concentrations: kanamycin 10 μg per ml, tetracycline 5 μg per ml, sucrose 50g per l. IPTG (Isopropyl β- d-1-thiogalactopyranoside) was added to the media to a final concentration of 1 mM when derepression of the LacI-repressed pTrc promoter was desired. Plasmids were extracted using the GeneJET Plasmid Miniprep Kit (Thermo Fisher Scientific).
Results
Rare alleles in multicopy replicons are prone to rapid loss
To experimentally quantify the effect of the replicon copy number on the fate of beneficial alleles, we studied the dynamics of an allele conferring resistance to the antibiotic kanamycin (nptII). The beneficial allele was introduced either on a polyploid (multicopy) plasmid or a monoploid chromosome in the model organism A. baylyi. Performing an experimental evolution experiment with serial transfers, we examined the effects of the initial allele frequency (i.e., the number of novel allele copies), the strength of the population bottlenecks, and the selection regime on allele dynamics (Fig 1A). We compare the results for three initial frequencies of cells containing the novel allele: f0 = 10−4 (ca. 105 cells, high), f0 = 10−6 (ca. 103 cells, moderate), and f0 = 10−7 (ca. 102 cells, low). The control of the initial frequency of cells containing the novel plasmid allele was achieved via natural transformation employing varying donor DNA concentrations (700, 7, or 0.7 ng/μl DNA). The novel nptII allele is introduced into an ancestral population that carries the stable model plasmid pTAD-R, which has a copy number of ca. 15. Homologous recombination between the donor DNA and the ancestral plasmid creates heterozygous cells carrying both the ancestral and the novel alleles, where the intracellular frequency of the novel allele is one plasmid copy out of 15 due to the one-hit kinetics of natural transformation (Fig 1A; ref. [28]). For the chromosomal allele, we controlled the initial allele frequency by mixing strains carrying either the novel or the ancestral allele at the abovementioned frequencies.
Discussion
Polyploidy allows for the coexistence of multiple alleles on a replicon–chromosome or plasmid–within a single cell, thereby creating intracellular genetic diversity. The establishment of alleles encoded in polyploid replicons is thus affected by drift and selection at two hierarchical levels: the collective of replicons within a cell and the collective of cells within the population. Intracellular processes–replication and segregation–generate an effect of stochasticity that ultimately increases genetic drift and interferes with the establishment of novel alleles.
Acknowledgments
We thank Daniela Kluger and Ian Dewan for their assistance in the experimental work. The authors thank Yiqing Wang, Shreya Vichare and Ishan Bhatt for critical comments on the manuscript, the entire Genomic Microbiology group and Stochastic Evolutionary Dynamics group for their help and discussions.
Citation: Garoña A, Santer M, Hülter NF, Uecker H, Dagan T (2023) Segregational drift hinders the evolution of antibiotic resistance on polyploid replicons. PLoS Genet 19(8): e1010829. https://doi.org/10.1371/journal.pgen.1010829
Editor: Ivan Matic, Institut Cochin, FRANCE
Received: February 2, 2023; Accepted: June 14, 2023; Published: August 3, 2023
Copyright: © 2023 Garoña et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: The source data underlying Figs 1 and 2 and Figs S1, S2, S3, S4, S5 and S6 are provided as a Source Data file. The code for our simulations is available online (https://github.com/mariosanter/multicopy-bottle-fixation).
Funding: AG is supported by the International Max Planck Research School (IMPRS) for Evolutionary Biology. TD is supported by the Leibniz Science Campus EvoLUNG. MS is a member of the International Max Planck Research School for Evolutionary Biology and gratefully acknowledges the benefits provided by the program. This work was furthermore funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) - project number 418432175 (to HU) and European Union (ERC) pMolEvol, grant number 101043835 (to TD). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
https://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1010829#abstract0
