MarA overexpression increases antibiotic resistance in population measurements

MarA’s role in activating downstream multidrug resistance genes has been studied extensively at the population level27,28,29,30,31,32. In this work we used carbenicillin, a bactericidal antibiotic that inhibits cell-wall synthesis35. We first measured the minimum inhibitory concentration of carbenicillin in E. coli MG1655 and in the same strain with a plasmid overexpressing MarA (Fig. 1b). Consistent with previous reports, overexpression of MarA increased antibiotic resistance36.

MarA expression is heterogeneous at the single cell level

Although inducible population-level resistance is well established, we wondered whether MarA expression is variable at the single cell level. Previous computational studies by our group have hypothesized that the feedback structure regulating MarA can produce stochastic MarA expression when the system is uninduced37. Motivated by these computational predictions, we experimentally measured the dynamics using a plasmid that reports MarA levels in the cell. To do this, we used a modified version of the marRAB promoter containing transversion mutations that inactivate the MarR binding sites in the operator, leaving the MarA binding site intact29. We fused this promoter to a cyan fluorescent protein gene (cfp) with an ssrA degradation tag to decrease the protein half-life and increase temporal resolution38. We conducted experiments with this plasmid in E. coli MG1655 (we refer to this strain as P marA -cfp). We note that MarR binding sites were only removed in the reporter plasmid; the chromosomal copy of the marRAB promoter remained unchanged. The promoter modification in the reporter was necessary to visualize CFP and allowed us to measure MarA independent of the action of MarR. In order to study dynamics and heterogeneity in MarA expression at the single cell level, we conducted time-lapse microscopy experiments with P marA -cfp. Within growing microcolonies we observed heterogeneous MarA expression that fluctuated over time (Fig. 1c and Supplementary Movie 1). Therefore, MarA expression is stochastic within single cell lineages.

MarA variability is correlated with survival in the presence of carbenicillin within an isogenic E. coli population

We next asked whether variability in MarA expression impacts survival under antibiotic treatment at the single cell level. Bacteria can transiently defend against antibiotic lethality by inducing the SOS response, which inhibits bacterial cell division but not elongation, enabling survival in the presence of lethal concentrations of antibiotics39. We exposed cells containing the MarA reporter P marA -cfp to lethal concentration of carbenicillin (50 μg/ml) on agarose pads and observed the impact on individual cells using time-lapse microscopy (Fig. 2a and Supplementary Movie 2). Cells lysis occurred rapidly after incubation with carbenicillin for a subset of cells in the population. As an indicator of cell death we used propidium iodide, which enters the cells and stains DNA if the membrane is depolarized40. Using P marA -cfp, we measured the initial fluorescence level of each cell at t = 0 mins. We then recorded the outcome of each cell after 400 mins of carbenicillin exposure (Fig. 2b). This duration, sufficient to kill a significant fraction of E. coli cells, allowed us to ensure that transient effects due to cell division time were not a factor in our analysis22. We primarily observed two outcomes: cell lysis, as indicated by propidium iodide staining and filamentation, where cells elongate but do not lyse. A small fraction (~10%) of cells neither stained with propidium iodide nor formed filaments and were excluded from subsequent analysis. Each cell outcome was assigned to the initial CFP value reflecting the MarA expression level in the cell at t = 0 mins. As expected from our time-lapse microscopy experiments (Fig. 1c), we observed a distribution of initial fluorescence levels corresponding to cell-to-cell variability in MarA expression (Fig. 2b). We also observed a heterogeneous response to carbenicillin. Interestingly, heterogeneous outcomes were correlated with MarA variability between isogenic cells, where cells that filamented were more likely to have high initial MarA levels (Fig. 2b).

Figure 2 Variability in MarA expression is correlated with a heterogeneous response to carbenicillin treatment. (a,c,e) Time-lapse microscopy images of (a) P marA -cfp, (c) P marA -cfp ΔmarRAB and (e) MarA-CFP in the presence of 50 μg/ml carbenicillin and 10 μg/ml propidium iodide. Cells were introduced onto agarose pads containing carbenicillin and propidium iodide at t = 0 mins and imaged over the course of 400 mins in two color channels. Cyan indicates CFP levels from the MarA reporter; red indicates the death marker propidium iodide. Supplementary Movie 2 shows additional details for the P marA -cfp strain. Note that in the MarA-CFP strain the localization patterns in CFP are due to binding of MarA to DNA. (b,d,f) Outcomes of individual cells after 400 mins of carbenicillin exposure, plotted versus CFP fluorescence at t = 0 mins for (b) P marA -cfp, (d) P marA -cfp ΔmarRAB and (f) MarA-CFP. Each blue dot corresponds to one cell, which has an outcome ‘lysed’ or ‘filamented’ and an initial fluorescence value. The number of cells exhibiting each outcome is listed on the x-axis. The mean ranks are statistically different for only the P marA -cfp strain (P < 0.01 by a Mann-Whitney rank sum test). Histograms and further details are provided in Supplementary Fig. 4. Full size image

To determine if variability in fluorescence levels was due to MarA expression itself, we introduced the same fluorescent reporter into a strain lacking the marRAB operon (we refer to this as P marA -cfp ΔmarRAB). We recorded initial fluorescence levels and cell outcomes in the presence of carbenicillin as before (Fig. 2c,d). Cells exhibited higher lysis rates following carbenicillin exposure than in the strain with the intact marRAB operon. CFP levels for P marA -cfp ΔmarRAB were lower than for P marA -cfp, as expected given the absence of MarA.

As a positive control, we also constructed a MarA-CFP protein fusion in order to produce a population with high, homogeneous expression of MarA. The translational fusion stabilizes MarA, increasing its half-life to ~30 mins (Supplementary Fig. 1), in contrast to ~1 min for wildtype MarA41. As a result, cells exhibited homogeneous fluorescence levels (Fig. 2e). It is important to note that the CFP levels for this strain do not report the same levels of MarA as those strains with P marA -cfp. Instead, the MarA-CFP strain has markedly higher levels of MarA than either the P marA -cfp or P marA -cfp ΔmarRAB strain due to the stabilized protein. When we exposed cells with MarA-CFP to carbenicillin, we observed a dramatic increase in the number of filamented cells relative to the P marA -cfp strain (Fig. 2f).

Filamented cells are able to regrow normally and are still susceptible to antibiotics

Are the filamented cells we observed following carbenicillin treatment able to resume growth after removal of carbenicillin? To test this, we used microfluidic chambers to trap cells while introducing and removing carbenicillin. Single cells were trapped in a microfluidic chamber and grown until the chambers were full. We then introduced a 90 min step of 50 μg/ml carbenicillin. Following this, we returned to conditions without the antibiotic, then later introduced a second step of carbenicillin (Fig. 3 and Supplementary Movie 3). As in our experiments on agarose pads with carbenicillin, we observed variability in MarA expression and heterogeneous responses, including both lysis and filamentation. Importantly, after carbenicillin was removed, the filamented cells were able to divide and regrow normally, suggesting the clinical relevance of transient antibiotic resistance. To confirm that these surviving cells were not resistant to antibiotics due to mutations or other non-transient mechanisms we introduced a second step of carbenicillin. We observed similar patterns of lysis and filamentation following this subsequent carbenicillin step, indicating that those cells that survived the first round of treatment were still susceptible to antibiotics.

Figure 3 Resistance to carbenicillin is transient and cells that survive resume normal growth. Time-lapse microscopy images of P marA -cfp ΔfliC cells growing inside a microfluidic chamber subjected to two sequential steps of 50 μg/ml carbenicillin. Cyan indicates CFP levels from the MarA reporter; red indicates the death marker propidium iodide, which was added at the same time as carbenicillin. Supplementary Movie 3 shows additional details. Full size image

MarA levels stochastically exceed a threshold that confers transient resistance to carbenicillin

We were next interested in understanding how dynamic, heterogeneous MarA expression impacts survival. We first quantified the dynamics of P marA -cfp within microcolonies. We observed that MarA levels fluctuate in individual cell lineages (Fig. 4a). We also quantified fluorescence levels in the P marA -cfp ΔmarRAB strain (Fig. 4b). Interestingly, the P marA -cfp ΔmarRAB strain still exhibited fluctuations in MarA expression, though fluorescence levels were reduced relative to P marA -cfp. These residual dynamics could be due to the action of the MarA homologs Rob and SoxS42, other regulatory mechanisms that interact with P marA 43, or dynamics intrinsic to the fluorescent reporter.

Figure 4 Level of MarA achieved by isogenic cells plays a key role in transient resistance to carbenicillin. (a) Representative fluorescence data extracted from a P marA -cfp microcolony. Gray traces show all cells within the microcolony, where branching indicates cell division. Green traces highlight representative lineages. (b) Representative fluorescence data for a P marA -cfp ΔmarRAB microcolony. (c) Histograms showing frequency (%) of cells with a given fluorescence value. Data comes from six microcolonies for P marA -cfp and three microcolonies each for P marA -cfp ΔmarRAB and P lac -cfp. (d) Autocorrelation of CFP signals for P marA -cfp (gray), P marA -cfp ΔmarRAB (magenta) and P lac -cfp (cyan). For each, we calculated the average autocorrelation for all cells within a microcolony. Error bars represent the standard deviation across replicates, which are described above. (e) Percentage of filamented P marA -cfp (gray) and P lac -cfp (cyan) cells as a function of the fluorescence threshold level. The percentage is calculated as the number of filamented cells divided by the total number of filamented and lysed cells. Full size image

We conducted control experiments to eliminate the possibility that something about CFP expression or the ssrA tag was responsible for increasing antibiotic survival. To achieve this, we constructed a reporter strain that was independent of MarA where we could induce similar CFP expression levels to the P marA -cfp strain. We refer to this control strain as P lac -cfp. We observed variation in CFP expression across cell lineages, likely due to the intermediate induction levels (Supplementary Fig. 2a). We also measured the distribution of fluorescence levels for the P marA -cfp, P marA -cfp ΔmarRAB and P lac -cfp strains (Fig. 4c). CFP fluorescence for P marA -cfp had a long tail of high fluorescence values. For P marA -cfp ΔmarRAB the distribution shape was similar, but the mean was slightly reduced and the tail of the distribution did not extend to CFP values that were as high as in the P marA -cfp strain. By design, the CFP levels for P lac -cfp were similar to or higher than those for P marA -cfp, but notably, the shapes of the distributions were different, suggestive of differing underlying dynamic processes44. To show that MarA levels and not a reporter artifact, were causing heterogeneity in antibiotic survival, we placed P lac -cfp cells on agarose pads containing carbenicillin and recorded cell lysis and filamentation outcomes as before. In contrast to results with P marA -cfp, we did not observe a correlation between higher fluorescence levels and filamented cells (Supplementary Fig. 2b), confirming the contribution of MarA to heterogeneous antibiotic survival.

We measured the average autocorrelation of the CFP signal for P marA -cfp, P marA -cfp ΔmarRAB and P lac -cfp strains and observed no dominant periodicity in expression of any of the CFP signals (Fig. 4d). However, we note that although the average autocorrelation of CFP is similar between strains and is not indicative of a periodic signal, this does not preclude the possibility that stochastic properties differ, as these effects may be obscured by an average. Similar experiments with the MarA-CFP overexpression strain showed fewer fluctuations in fluorescence levels and slower dynamics, as expected due to the stabilized protein (Supplementary Fig. 3).