Plasmid copy number noise in monoclonal populations of bacteria

Plasmids are extra chromosomal DNA that can confer to their hosts’ supplementary characteristics such as antibiotic resistance. Plasmids code for their copy number through their own replication frequency. Even though the biochemical networks underlying the plasmid copy number (PCN) regulation processes have been studied and modeled, no measurement of the heterogeneity in PCN within a whole population has been done. We have developed a fluorescent-based measurement system, which enables determination of the mean and noise in PCN within a monoclonal population of bacteria. Two different fluorescent protein reporters were inserted: one on the chromosome and the other on the plasmid. The fluorescence of these bacteria was measured with a microfluidic flow cytometry device. We show that our measurements are consistent with known plasmid characteristics. We find that the partitioning system lowers the PCN mean and standard deviation. Finally, bacterial populations were allowed to grow without selective pressure. In this case, we were able to determine the plasmid loss rate and growth inhibition effect.

Figure 1

(Color) The experimental setup is built on an inverted microscope. Laser light is shaped to a constant intensity disk of $\sim 20\mu \text{m}$ diameter in the field of view by a spatial light modulator (SLM). Fluorescent light is collected by the same objective and separated on green and orange channels through a combination of dichroic and emission filters. A subregion of the field of view (a $\sim 10\mu \text{m}$ disk) containing the channel through which the bacteria pass is focused on the avalanche photodiodes. TTL pulses from the avalanche photodiodes are fed to a counting acquisition board in a computer. A bacterium passing through the field of view causes an increase in the measured fluorescence intensity.

Figure 2

(Color) [(a) and (b)] Temporal signal from the counting module, respectively, for the green and for the orange fluorescences. An abrupt increase is perceived when a bacterium passes through the detection field of the photon counting module. The peak and baseline of each event are spotted in blue and black, respectively. (c) Fluorescence scatter plot of three different populations. Bacteria bearing the green expression system on the chromosome without any plasmid is in green. Bacteria with the orange expression system on the same location as the previous strain also without a plasmid is in orange. The original strain bearing no fluorescence is in black. The leakage coefficients $\alpha$ and $\beta$ and the normalization factor $a$ (see Sec. 3C) are deduced from the difference in mean fluorescence of those three populations. $\alpha$ could not be measured as it was too small $\left(<1\mathrm{\%}\right)$ and $\beta$ was estimated to $0.1674\pm 0.0005$. $a$ was calculated to be $0.58\pm 0.04$, which corrects for differences in optical pathways, fluorescent excitation, emission efficiency, and differences in expression. [(d) and (e)] Population histograms of green and orange fluorescence shown in (c). (f) Normalized histograms of EGFP and mOrange expressed from a mini-colE1 plasmid.

Figure 3

Mean expression from the mini-F plasmid normalized to the expression from the chromosome as a function of the number of mOrange constructs in the mini-F plasmid. The line is a linear fit with a slope of $1.03\pm 0.02$.

Figure 4

Mean (a) and noise (b) in PCN for the mini-F (squares), the mini-ColE1 (triangle, inset), the $\text{mini-R}1\text{−}pa{r}^{-}$ (open circle), and the $\text{mini-R}1\text{−}pa{r}^{+}$ (closed circle) as a function of temperature. The error bars are standard deviations obtained from multiple experiments. Error bars were not plotted whenever they were smaller than the data point size.

Figure 5

Histograms of the orange fluorescence $\left(F_O\right)$ in logarithmic scale for the $\text{mini-R}1\text{−}pa{r}^{-}$ (a) and the $\text{mini-R}1\text{−}pa{r}^{+}$ (b) at different generation times. In black (plain), the plasmids are in the presence of pressure selection. The red (dotted) and blue (dashed) histograms of the population are the fluorescence distributions after 54 and 99 generations in the medium without antibiotics, respectively.