Earlier in these pages I gave a brief background of what I've been doing for the past year. I've been interested in how differences in mRNA production and differences in holin structure led to variability in lysis timing. Max Delbruck was the first to consider this question; he looked at variation in the number of babies a single phage produces and found a great deal of variation in this important life history trait. Delbruck's method, diluting a suspension of infected bacteria such each dilution contained on average less than one bacterial cell, and then plating all the (hundreds of) dilutions, is especially onerous, and perhaps explains why his work was not followed up.
(i.e. production rate, and observe the changes in Moreover, his work leaves open the questions of what causes the variation and whether it is evolutionarily significant. My work follows up on Delbruck's, except that I chose to look at a different, but closely related life history trait, lysis time. Earlier work has shown that there is a direct correlation between lysis time and burst size: the longer the phage waits to lyse the cell, the more babies it can produce. So it makes a good proxy for burst size, as well as an interesting life history trait in its own right. Plus, we now have the genetic techniques to manipulate the phage's genome and dissect the causes of variation: to wit, the ability to manipulate the holin protein structure and to alter the strength of the promoter that controls holin production. All we need is a way of observing lysis time for individual cells. Enter the microscope-mounted perfusion chamber! Here is the setup:
The way this works is you place a glass cover slip on the bottom, a cell-binding agent (poly-lysine) and some E. coli infected with lysogenic phage, then place another cover slip on top. The cells form a single layer on the bottom cover slip. The whole unit is placed on a heating platform, under a normal light microscope, and tubes are attached to allow a flow of nutrient broth thru the chamber. The heating platform generates a heat spike, inducing the phage to begin the lysis process. With a microscope mounted camera, I filmed the bound cells following the heat spike until they lysed, then recorded the time of lysis for each cell.
Given are the variation in lysis time for the wild type (JJD3), the most variable altered holin sequence genotype (JJD9) and the most variable altered promoter genotype (SYP028). This histogram (below) clearly shows the greater variability (standard deviation, SD, in lysis time for the latter two genotypes.
Whether the observed lysis time stochasticity is evolutionarily significant remains to be determined; however it is conceivable that selection can favor genotypes with greater or lower levels of phenotypic stochasticity depending on the circumstances. Future experiments will address (1) whether variation in lysis time stochasticity translates into variation in fitness; (2) whether genotypes expressing greater or lower levels of lysis time stochasticity can be selected for; (3) whether similar patterns of stochasticity exist for different phage species.