Phage Biology

For many years, our students have been interested in the process of phage infection under
conditions more like those in nature. Our early work and that of others was briefly summarized in our chapter Effects of Host Physiology and Growth Conditions in the 1994 T4 book, and the next talks will report on their recent experiments with various T-even phages under anaerobic conditions.

We have also done some additional work exploring the infection of cells in "stationary phase", particularly looking at their ability to form stable infective centers that can go on to make a plaque when plated using a lawn of exponentially-growing cells. These experiments build on the Kolter lab’s studies of bacterial physiology under conditions stretching for many weeks beyond exponential phase in rich medium. We used their strain ZK126, which is wild type with regard to the stationary-phase sigma factor (in contrast to the attenuated version that has developed in many lab K12 strains) as well as a derivative, ZK1000, in which the sigma S is missing. We followed the ability of T4 to infect cells that have been "growing" for up to 20 days after innoculation into LB or M9, using a total of 107 phage in all cases, so most cells are uninfected. We find that the T4 are able to adsorb well throughout this period; we saw only 0.1-1% unadsorbed phage. At 48 hours, all of the infected cells could make infective centers that were fully stable for 4 hours; half could still develop into infective centers after 24 hours. Both the ability to form infective centers and their stability gradually decreased after that, but when infected after 4-10 days 10 % could form infective centers, with over one percent even at 20 days, and these were stable for at least 4 hours. We have named this unique form of T4 infection "hibernation". We have carried out a few protein labeling/ gel electrophoresis experiments (under high MOI conditions) to look at what happens when nutrients are added to such cells in hibernation mode. They show that all of the stationary-phase cells can be infected and that initially the same few lag-phase proteins seem to be made in both infected and uninfected cells. However, before two hours the uninfected cells are making the usual plethora of proteins, while the infected cells are making a typical late pattern of phage proteins and a small burst of phage is produced. In the absence of the stationary-phase sigma factor, the cells do not seem to go into hibernation mode in the same fashion. This mode of infection may help explain how the very virulent T-even phages coexist with their hosts in the wild; clearly, far more work is needed here.

We are also interested in the mucoid P. aeruginosa from cystic fibrosis patients. We found that the Pyophage mixture from Tbilisi would infect 17 of 18 such strains that we got from Steve Lory in Seattle, and that it was surprisingly easy to isolate phage against such strains – every liter of local sewage gave us phage against whichever of these strains we added! We have been exploring various properties of several of the new phages that we isolated in this fashion, which we will present. We are looking forward to working with Kalai Mathee on developing ways to effectively test some of these phages for their ability to infect biofilms, as well as working with other colleagues to then test biofilm infectivity of phages that appear potentially useful for treating CF patients.

Clearly, there are infinite interesting possibilities for exploring phage infection under environmentally interesting conditions, and we strongly encourage our colleagues to work out ways to explore some of them as group or individual student projects. As discussed in a recent exchange through Steve Abedon’s phage ecology website, some very interesting procedures and experiments are much easier to carry out in timely, sterile and inexpensive fashion using mouth pipeting than with any of the devices we have found. However, we are being pushed hard to totally abandon that practice. Over the next days, we would very much appreciate input as to how others deal with this issue in their institutions.

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Last Updated: May 29, 2008

The Evergreen State College

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