Tracking bacterial gene expression with firefly luciferase
Because gene expression within a cell population fluctuates randomly, investigators must monitor individual cells if they want to follow the activity as genes switch on and off. But doing so with standard GFP or its derivatives, a commonly used approach, is difficult. GFP is almost undetectable at the single-molecule level and takes hours to mature, which makes it impossible to follow rapidly changing processes.
Now researchers from Iowa State University in Ames have come up with a different method. They found a way to introduce firefly luciferase into bacteria as a chemiluminescent reporter. Firefly luciferase is much brighter than the bacterial luciferase sometimes used for chemiluminescence studies. The process requires the introduction of the substrate luciferin into the cells, because the reaction of the substrate with luciferase in the presence of oxygen and ATP results in light.
Researchers have developed a way to use firefly luciferase — which is much brighter than bacterial luciferase — as a chemiluminescent reporter in bacteria. On the left are bright spots showing chemiluminescence from individual bacteria. On the right is a transmitted-light image of the bacteria, confirming that the emitted light is localized to the individual cells. Courtesy of Edward S. Yeung, Iowa State University.
The technique offers detection without a time delay and at nearly the best possible level, said research team leader and chemistry professor Edward S. Yeung. “The sensitivity approaches the detection of a single enzyme, which is the ultimate limit for any detection scheme.”
However, the challenge is getting the luciferin into the bacteria. The pores in bacterial cell walls are too small for the charged luciferin molecules to fit through. The pores can be enlarged by putting the cells in an acidic solution, but even then only neutral molecules can cross, and the resulting emission is too weak to allow single-molecule detection.
Yeung said that the researchers accidentally observed that dehydration made the cell wall porous enough to introduce luciferin. In fact, it did so without harming the cells, which the investigators verified by rehydrating them.
Along with graduate student Yun Zhang and professor of veterinary medicine Gregory J. Phillips, Yeung used the chemiluminescence technique to perform quantitative studies of gene expression in two bacterial lines. They engineered BL21 and XLU102 E. coli strains to express firefly luciferase. The first strain was otherwise normal, while the second was defective in its handling of the sugar arabinose.
They imaged the bacteria with a Nikon microscope, associated lenses and an electron-multiplying microchannel plate-coupled CCD from Princeton Instruments of Trenton, N.J. The system was calibrated by recording the light generated from standard solutions of firefly luciferase in sequential frames over a 30-s exposure.
They then took a small sample from the growing cell cultures at hourly intervals, dehydrated the cells and recorded the chemiluminescence.
The investigators saw a clear difference in the two cell lines and were able to map the spatial distribution of luciferase within a given cell. Cells express proteins within certain subcompartments, and the observed luminescence fits this general phenomenon. The studies revealed that the cells seemed to continue to divide during a quiescent period and that the expressed firefly luciferase seemed to be transported to certain locations over the cells’ growth period. Yeung noted, however, that the team could not conclude where exactly the luciferase was expressed in the bacteria because of inadequate spatial resolution.
He said that the next step would be to use the scheme to follow metabolism in bacteria as a function of such external stimuli as oxygen, carbon dioxide, temperature and drugs. “ATP levels change in all cells when stressed. We will simply measure the changing chemiluminescence intensities.”
Analytical Chemistry, Feb. 1, 2008, pp. 597-605.
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