Measuring the Rate of Conjugal Plasmid Transfer in a Bacterial Population Using Quantitative PCR

Abstract

Horizontal transfer of genes between species is an important mechanism for bacterial genome evolution. In Escherichia coli, conjugation is the transfer from a donor (F(+)) to a recipient (F(-)) cell through cell-to-cell contact. We demonstrate what we believe to be a novel qPCR method for quantifying the transfer kinetics of the F plasmid in a population by enumerating the relative abundance of genetic loci unique to the plasmid and the chromosome. This approach allows us to query the plasmid transfer rate without the need for selective culturing with unprecedented single locus resolution. We fit the results to a mass action model where the rate of plasmid growth includes the lag time of newly formed F(+) transconjugants and the recovery time between successive conjugation events of the F(+) donors. By assaying defined mixtures of genotypically identical donor and recipient cells at constant inoculation densities, we extract an F plasmid transfer rate of 5 × 10(-10) (cells/mL · min)(-1). We confirm a plasmid/chromosome ratio of 1:1 in homogenous F(+) populations throughout batch growth. Surprisingly, in some mixture experiments we observe an excess of F plasmid in the early saturation phase that equilibrates to a final ratio of one plasmid per chromosome.

Analysis of bacterial genomes from an evolution experiment with horizontal gene transfer shows that recombination can sometimes overwhelm selection

“…Indeed, more recent studies have found that F plasmid-mediated conjugation readily occurs in E . coli at the 120 rpm shaking speed of the STLE, albeit under different conditions [ 32 , 33 ]. We found sequencing reads that map to the entire F plasmid in both Ara–3 clones, but not in any other clones we sequenced ( S4 Fig ).…”

Dissecting the effects of antibiotics on horizontal gene transfer: Analysis suggests a critical role of selection dynamics

“…However, the detection must be sensitive enough to capture events occurring at low frequencies. Nucleic acid measurements, such as qPCR, can also be used in measuring the conjugation efficiency in certain systems [107]; this type of quantification does not require transconjugant enrichment (e.g. by selection), and could circumvent biases introduced by growth.…”

The Detailed Bactericidal Process of Ferric Oxide Nanoparticles on E. coli

“…The qPCR assays were used to quantify bacterial amount at every time point, using the genetic locus TolC of the bacteria [ 44 ] and Power SYBR ® Green Master Mix (Applied Biosystems, Foster, CA, USA) in an Applied Biosystems 7300 (Applied Biosystems, Foster, CA, USA). Lysed and frozen aliquots of the bacteria (2 μL) were used as templates for the qPCR reactions (20 μL).…”

“…The qPCR conditions were as follows: an initial denaturation at 95 °C for 30 s; 40 cycles of 95 °C for 5 s; 60 °C for 60 s. We set the following mathematical model to analyze the effect of ferric oxide nanoparticles on bacterial growth. As batched bacterial cell cultures were assayed, we assumed a logistic bacterial growth, as described previously [ 44 , 45 ]. The logistic model states that the number of cells in a state as a function of time ( N ′) is the product of the growth rate ( ψ ), the number of cells currently in that state ( N ), and the saturation limit of the media ( K ).…”