Monday, July 14, 2008

On the shoulders of giants

I assume this is a reference to the Isaac Newton quote "If I have seen farther than others, it is because I have stood on the shoulders of giants.". But my friend Megan passed this along to me, which is essentially a call to post about a classic paper in your field and the contributions it's made.

The papers which have had the most direct effect on my science have been relatively more recent, but thankfully, I do know some useful older ones as well. If I were feeling more ambitious, I'd tackle Sewall Wright's 1932 paper which laid out shifting balance theory, but that's not going to fit into my current time scheme. So I'll instead go with Lederberg and Lederberg's March 1952 classic Replica Plating and Indirect Selection of Bacterial Mutants in the Journal of Bacteriology

Though it's hard to think of it at the moment, at the time this paper was written there was controversy over the origin of mutations. Some people felt that mutations were always arising spontaneously. Others felt that mutations which conferred adaptation to a specific condition would be brought about by introduction of that condition. For example, if a cell were subjected to an increased salt concentration, mutations which enabled it to deal with a high salt concentration (maybe changes in ionic transporters, changes in the internal salt concentration, changes in detoxification machinery, etc) would be induced, and thus occur more frequently than they would in a cell not subjected to salt stress. Thought this may seem strikingly teleological, I feel it's important to remember that this was before the double helix nature of DNA had been demonstrated, so it was a time in which biology was even more of a black box than it is currently.

The Lederbergs devised a simple experiment to test this idea. They grew bacteria on agar plates without the presence of an antibiotic that the strain was sensitive to. They then stamped this master plate onto a sterile piece of velveteen, so some of the cells adhered to the pile. This velveteen was then stamped onto a number of fresh plates containing the antibiotic the cells were known to be sensitive to. If the mutations conferring resistance happened before exposure to the antibiotic, resistant colonies would form in the same location on the fresh plates, because they would have been derived from a resistant ancestor on the master plate. Conversely, if the mutations were induced by the presence of the antibiotic, there would be no correlation in location of the resistant colonies from one plate to another. The results clearly showed that the locations where resistant colonies grew were consistent across the replica plates made from a given master plate, showing that the mutations responsible for the adaptation arose in an environment in which they would not have been expected to be advantageous. This paper also details the equivalent experiment involving bacterial resistance to a virus which infects the wild type--again, resistance crops up in the same locations repeatedly, indicating that it is derived from changes which occur before exposure to the virus.

This paper also outlines the use of sterile velvet for replica plating--a technique I myself have used repeatedly in the lab to screen for certain types of mutations and/or genetic engineering. In general, it's fairly easy to screen most traits in one direction--if you want to find which bacteria are resistant to a given antibiotic, you put the antibiotic in their growth medium, and anything that grows will resist it. Sometimes, though, you want to select for the cells which are sensitive to the antibiotic, or which require the addition of a certain chemical in order to grow, or the like. Replica plating provides an efficient way to screen hundreds of colonies for these types of changes--make a master plates in the permissive environment (without the antibiotic, with any possible required nutrient, etc), and make replica plates on both the permissive and the strict environment (when the antibiotic is present, when a given compound is missing, etc.). Look for colonies which grow in the permissive environment but which don't in the sensitive one. There you go. To be more sure of yourself, you'll generally repeatedly test that it has the property you're looking for, but the odds are pretty good that it does, and it's a lot faster than other means of finding such negative properties.

So, thank you, Drs. Lederberg. this paper of yours not only established the importance of mutation prior to exposure to an environmental challenge, but also outlined a handy lab technique I've made repeated and systematic use of in my own experiments.