Dynamical determinants of inducible gene expression in a single bacterium

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Original Entry: Dariela Almeda - AP 225, Fall 2009.

Article Authors: Thuc T. Le, Thierry Emonet, Sebastien Harlepp, Calin C. Guet, and Philippe Cluzel.

Keywords

Efflux pump, tetracycline, gene expression.

Summary

The article focuses on characterizing the dynamics of an acrAB-TolC efflux pump in a single E. coli bacterium using a tet-promoter (short for the antibiotic tetracycline). They chose this specific promoter because it is known that tetracycline is a substrate of this efflux pump. Bacterial efflux pumps are known to have significant effects on antimicrobial resistance due to their ability to pump out antibiotics from bacterial cells. In order to carry out their experiments, they first had to find a solution of how to monitor the transcriptional activity of a single bacterium. Their solution to this problem consisted of attaching the bacteria to a poly-L-lysine coated coverslip in order to prevent its growth but remain preserve the bacteria's ability to respond to chemical cues. Moreover, they used a double-plasmid system where they transformed the bacteria with 2 plasmids. One plasmid, called MS2-GFP, expressed a phage coat protein fused with green fluorescent protein (GFP) and regulated by a Lac-promoter. The second plasmid, called ms2-RNA, was regulated by the tet-promoter. When the bacteria were exposed to the antibiotic, the acrAB promoter increased sharply and then plateaued as seen in Figure 1. The concentration of the antibiotic was then varied in 10 single cells and the synthesis rate of ms2-RNA was similar for all concentrations as seen in Figure 2. To explain these results, the authors concluded that either synthesis or degradation of RNA was causing this phenomenon. They added the antibiotic rifampicin in order to stop the transcription of ms2-RNA and observed that the RNA degradation was independent of the concentration of tetracycline used. Thus, they deduced that RNA synthesis was responsible for the pulsating transcriptional response. The article also presents a model of the transcriptional activity in a single cell. Some of the assumptions include RNA synthesis being regulated only by concentration of inducer (tetracycline), equal initial intracellular and extracellular inducer concentrations, and a constant intracellular inducer concentration that increases with time. Their theoretical results agreed with their experimental results. Finally, they also repeated the experiments with a mutant bacterial strain that had a defective efflux pump. The concentration of ms2-RNA was higher in the mutant cells than in the wild type after being subjected to equal inducer concentrations. This suggests the efflux pump regulates the intracellular concentration of inducer which in turn regulates transcription activity.

Soft Matter Connection

The importance behind the multidrug resistant efflux pumps lies on the transport of antibiotics through the bacterial cell membranes and in and out of the cell. The results from the article suggest that antibiotics are responsible for modulating gene expression levels in bacteria. The transport of drugs in bacterial cells is mainly mediated by concentration gradients across the cell membrane due to the simplicity of the bacterial cell structure. It would be interesting to compare the energetic costs of pumping drug in and out of the cell and of the drug passing through the cell membrane. Another point that was mentioned in the article was how the halt of cell proliferation when bound onto the poly-L-lysine coated substrate is still not completely explained. Quantifying the mechanical forces on each individual cell and understanding how the poly-L-lysine interacts with the cell surface/membrane can shed light on this problem. Maybe the adsorption onto the poly-L-lysine has an effect on membrane proteins that signal the cell stop proliferation.

Fig1 da.png

Figure 1. acrAB promoter activity induced with 400 ng/ml of tetracycline.

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Figure 2. tet promoter activity of ten single bacterial cells induced at (A) 200 ng/ml of tetracycline, (B) 400 ng/ml, and (C) 1 ug/ml. (D) represents the average ms2-RNA concentration of ten cells. No induction (light gray), 200 ng/ml tetracycline (gray), 400 ng/ml (dark gray), and 1 mg/ml (black).