Figure 4.

Effect of tetracycline treatment on the autofluorescence, protein synthesis and viability of E. coli cells. E. coli 7705035 cells were treated with a range of tetracycline concentrations for 3 hours of the exponential growth. The MIC of this strain is 1 mg/L of tetracycline. Autofluorescence (a) and cell viability were assessed using AF633H (b) and TOPRO-3 (c) dyes and flow cytometer. As a control for staining of dead cells, cells were killed by incubation at 65 °C for 30 min. The impact of tetracycline on protein synthesis was assessed using lacZ (d) and cspA (e) promoters which were fused to the gene coding for the fast folding green fluorescent protein (GFP). cspA is known to be induced by protein synthesis inhibiting antibiotics, while lacZ should be repressed. Exponentially growing E. coli cells carrying plasmids with these reporter fusions, as well as promoter-less control plasmid, were incubated with IPTG and a range of tetracycline concentrations for 3 hours. Background autofluorescence was removed by subtracting the promoter-less autofluorescence. GFP fluorescence and FSC of each cell was monitored with the flow cytometer and cell autofluorescence or GFP fluorescence was normalized by the FSC. Values represent the mean (+/−standard error) of the median values of three independent experiments.