Quantifying plasmid dynamics using single-cell microfluidics and image bioinformatics

Hernandez‐Beltran, J. Carlos R.; Rodríguez-Beltrán, Jerónimo; Millán, Álvaro San; Peña‐Miller, Rafael; Fuentes-Hernández, Ayari

doi:10.1016/j.plasmid.2020.102517

Cited by 6 publications

(12 citation statements)

References 96 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In particular, here we used Escherichia coli MG1655 carrying pBGT, a non-transmissible multi-copy plasmid used previously to study plasmid dynamics and drug resistance evolution (San Millan et al . 2016; Rodriguez-Beltran et al 2018; Hernandez-Beltran et al 2020). Briefly, pBGT is a ColE1-like plasmid with ∼19 plasmid copies per cell, lacking the necessary machinery to perform conjugation or to ensure symmetric segregation of plasmids upon division.…”

Section: Resultsmentioning

confidence: 99%

“…We validated our model using a well characterized model plasmid, pBGT (San Millan et al 2016; Rodriguez-Beltran et al 2018; Hernandez-Beltran et al 2020). By analyzing growth kinetics of the plasmid-bearing and the plasmid-free strains we estimated the maximal growth rates of both strains, as well as the fitness cost associated to plasmid bearing and the fitness advantage of the plasmid bearing cells for different antibiotic concentrations.…”

Section: Discussionmentioning

confidence: 99%

“…We validated our model using a well characterized model plasmid, pBGT. 21, 34, 35 By analyzing growth kinetics of the plasmid-bearing and the plasmid-free strains we estimated the maximal growth rates of both strains, as well as the fitness cost associated to plasmid bearing and the fitness advantage of the plasmid bearing cells for different antibiotic concentrations. By numerically solving our model with parameters obtained from the experimental data, we found that selection is necessary for persistence of costly plasmids.…”

Section: Discussionmentioning

confidence: 99%

See 2 more Smart Citations

Segregational instability of multicopy plasmids: a population genetics approach

Pina

Hernandez‐Beltran

Siri‐Jégousse

et al. 2022

Preprint

View full text Add to dashboard Cite

Plasmids are extra-chromosomal genetic elements that encode a wide variety of phenotypes and can be maintained in bacterial populations through vertical and horizontal transmission, thus increasing bacterial adaptation to hostile environmental conditions like those imposed by antimicrobial substances. To circumvent the segregational instability resulting from randomly distributing plasmids between daughter cells upon division, non-transmissible plasmids tend to be carried in multiple copies per cell, with the added benefit of exhibiting increased gene dosage. But carrying multiple copies also results in a high metabolic burden to the bacterial host, therefore reducing the overall fitness of the population. This trade-off poses an existential question for plasmids: What is the optimal plasmid copy number? In this manuscript, we address this question using a combination of population genetics modeling with microbiology experiments consisting of Escherichia coli K12 bearing a multi-copy plasmid encoding for bla TEM-1 , a gene conferring resistance to β -lactam antibiotics. We postulate and analyze a Wright-Fisher model to evaluate the interaction between selective pressure, the number of plasmid copies carried by each cell, and the energetic cost associated with plasmid bearing. By numerically determining the optimal plasmid copy number for constant and fluctuating selection regimes, we show that the stability of multi-copy plasmids is maximized at intermediate plasmid copy numbers. We conclude by arguing that plasmid copy number is a highly optimized evolutionary trait that depends on the rate of environmental fluctuation and balances the benefit between increased stability in the absence of selection with the burden associated with carrying multiple copies of the plasmid.

show abstract

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Segregational instability of multicopy plasmids: a population genetics approach

Pina

Hernandez‐Beltran

Siri‐Jégousse

et al. 2022

Preprint

View full text Add to dashboard Cite

show abstract

“…After 12 h of incubation, we observed that ICE Bs1 transfer was initiated in a few donor bacteria with strong mKate2 fluorescence scattered through the biofilm, while most donor cells showed low fluorescent red signal (Figure 2A). This discrepancy between the fluorescence of various donor cells is likely due to the activation of ICE Bs1 and its subsequent replication, which would increase the number of mKate2 copies (37, 38). Four hours later, those initial donors had transferred ICE Bs1 to neighboring cells, forming clusters of mKate2+ cells (Figure 2A).…”

Section: Resultsmentioning

confidence: 99%

Second-generation transfer mediates efficient propagation of ICEBs1 in biofilms

Bourassa

Jeannotte

Lebel-Beaucage

et al. 2022

Preprint

View full text Add to dashboard Cite

Horizontal gene transfer (HGT) by integrative and conjugative elements (ICEs) is an important mechanism in the spread of antibiotic resistance genes. However, little is known about the spatiotemporal dynamic of ICEs propagation in bacterial biofilms, which are multicellular structures ubiquitous in the natural and clinical environment. Using a fluorescently marked ICEBs1, we report here that its propagation in biofilms is favored in recipient cells expressing the biofilm matrix. Also, conjugation appears restricted to clusters of bacteria in a close neighborhood in which a high level of ICEBs1 transfer occurs. These conjugative clusters are heterogenously distributed in the biofilm, forming close to the air-biofilm interface. Importantly, we established that transconjugant cells are the main contributors to ICEBs1propagation in biofilms. Our findings provide a novel spatiotemporal understanding of ICEs propagation in biofilms, which should have an important role in our understanding of horizontal gene transfer in relevant settings.

show abstract

“…54 β -lactam resistance genes are generally located on plasmids and, in particular, TEM-1 has a plasmid origin, with more than two-hundred TEM β -lactamase variants descending from this allele recorded. 55 We denote the strain carrying this wellcharacterized, 45,56 non-conjugative, and multicopy plasmid as MG/pBGT (average copy number=19.12, s.d.= 1.53; Figure 1A-C). 45 As a control, we used a fluorescently tagged strain carrying a chromosomally encoded bla TEM-1 , which we term MG:GT.…”

Section: Environmental Modulation Of Pcn Distributions In Bacterial P...mentioning

confidence: 99%

Antibiotic heteroresistance generated by multi-copy plasmids

Hernandez-Beltran

Rodríguez-Beltrán

Aguilar-Luviano

et al. 2022

Preprint

View full text Add to dashboard Cite

Heteroresistance - in which a clonal bacterial population contains a cell subpopulation with higher resistance to antibiotics than the main population - is a growing clinical problem that complicates susceptibility determination and threatens therapeutic success. Despite the high prevalence of heteroresistance in clinical settings, the underlying genetic mechanisms that stably maintain heterogeneous bacterial populations are poorly understood. Using fluorescence microscopy, single-cell microfluidics, and quantitative image analysis, we show that random replication and segregation of multicopy plasmids produce populations of bacterium Escherichia coli MG1655 in which cells with low- and high-plasmid copy numbers stably co-exist. By combining stochastic simulations of a computational model with high-throughput single-cell measurements of blaTEM-1 expression, we show that copy number variability confers the bacterial population with transient resistance to a lethal concentration of a beta-lactam antibiotic. Moreover, this surviving, high plasmid copy minority is capable of regenerating a heterogeneous bacterial population with low and high plasmid copy numbers through segregational instability, rapidly alleviating the fitness burden of carrying large numbers of plasmids. Our results provide further support for the tenet that plasmids are more than simple vehicles for horizontal transmission of genetic information between cells, as they can also drive bacterial adaptation in dynamic environments by providing a platform for rapid amplification and attenuation of gene copy number that can accelerate the rate of resistance adaptation and can lead to treatment failure.

show abstract

Quantifying plasmid dynamics using single-cell microfluidics and image bioinformatics

Cited by 6 publications

References 96 publications

Segregational instability of multicopy plasmids: a population genetics approach

Segregational instability of multicopy plasmids: a population genetics approach

Second-generation transfer mediates efficient propagation of ICEBs1 in biofilms

Antibiotic heteroresistance generated by multi-copy plasmids

Contact Info

Product

Resources

About