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Single-molecule fluorescence polarization and "magnetic tweezers" could help scientists get a grip on viruses known as bacteriophages which attack and kill bacteria. The research could lead to a resurgence of interest in a potent treatment for infection that began in the first half of the twentieth century but was overshadowed in western medicine by the advent of antibiotics. The same research could also lead to new insights into how to combat viruses that infect people too, including herpes and adenoma viruses. Bacteriophages, are viruses that infect bacteria, multiply within and kill them. The name literally means bacterium eater. Because the phages themselves contain a fluxional genetic code they can adapt quickly to overcome any resistance a particular pathogenic bacterium may evolve and so can keep up with emerging resistance in a way that no passive antibiotic can. In the face of deadly bacteria such as Escherichia coli O157, multiple-resistance Staphylococcus aureus (MRSA) and Clostridium difficile, there is renewed interest in this alternative to antibiotics. As with many other viruses, the lifecycle of a bacteriophage includes a self-assembly stage in which a powerful molecular motor must pack the genome into the virus' preformed protein shell, the capsid. "Many viruses that infect humans such as the herpes, and adenoma viruses pack their DNA by a similar mechanism," Carlos Bustamente of the University of California, Berkeley, told SpectroscopyNOW. But, how any virus achieves this intricate feat has remained a subject of debate for many years. The most common explanatory mechanism involved the DNA being coiled like so much spaghetti on a fork by rotation of the motor relative to the capsid, "a sort of 'molecular roto-rooter,' Bustamente adds. Bustamente and colleagues were doubtful of this theory. They think they now have the answer to genetic packing for the bacteriophage Bacillus subtilis phi29. The team trapped the end of a single viral capsid using antibodies to glue it to farthest from the capsid opening. They then stretched out its DNA by attaching a magnetic bead to one end and using a magnet to draw it out. A fluorescent dye was then used as the flag for single-molecule fluorescence polarization spectroscopy studies of how the virus then packed the magnetically unravelled DNA. The researchers explain that if the motor rotated to package the DNA, then the fluorescence dye would produce as sine wave-like fluctuation in intensity. However, they did not observe such behaviour. Indeed, a mathematical analysis of the fluorescence pattern confirmed that there was no rotational component to the process of which the team is 99% certain. So, how does the phage wrap up its DNA? The team suggests that their analysis is compatible with a recently proposed non-rotating model in which the ring of the enzyme ATPase alternately compresses and extends to draw the DNA into the capsid. Further testing will be needed to confirm the validity of this model, but in the meantime research into phage therapy should continue with a view to eating bacteria. "Clearly, understanding the intricate mechanism of these motors by using single molecule studies, will eventually lead to the development of drugs that can block the motor," Bustamente told us, "we could arrive at therapies against these pathogens."
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Bustamente, unravelling secrets of phage packing
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