Studying the nature of T4 resistance

In your experiments to isolate T4-resistant E. coli, you first added phage to a culture. 

After about 25 minutes, there was an initial dramatic drop in absorbance.  

Over a period of 24-72 hours, some of the cultures became cloudy again! 

• Why don't all cultures show growth?
• Would it help if we waited even longer?
• Would you expect to find phage in the cultures that are growing?

Our next step is to determine whether resistance to T4 phage is stable, inheritable and specific for T4, i.e. are T4 resistant cells resistant to viruses unrelated to T4.

There are many types of coliphage, that is viruses that can infect E. coli.  We will use the T-odd phage T1, which is actually unrelated to T4. 

We begin our studies by generating a new culture of T4 resistant E. coli; we will start the culture from a single colony. 

Label this culture T4R-1, also grow up a culture of wild type E. coli. 

Divide the T4R-1 culture into two; take 2 ml of the original culture and add 3ml of fresh LB; do the same for the wild type culture. 

Experiment: 

• Inoculate wild type culture 1 with 10⁸ T4 PFUs
• Inoculatewild type culture 2 with 10⁸ T1 PFUs.
• Inoculate T4R-1 culture 1 with 10⁸ T4 phage/ml.
• Inoculate T4R-1 culture 2 with 10⁸ T1 phage/ml.

• What did the experiment using culture 1 control for?
• What did the experiment using culture 2 control for?
• What did the experiment using T4R-1 culture 1 tell you?
• What did the experiment using T4R-2 culture 2 tell you?
• all of your original TR cultures stably resistant to T4?
• What are the possible results from this experiment, assuming that both T1 and T4 phage stocks were active? 

Phage recognize their targets through interaction with molecules on the bacterial cell surface.

The initial recognition event involves the tail fibers.

If the phage finds the "right" type of bacteria, interactions with baseplate lead to irreversible binding.

Binding of the base plate leads to the injection of the phage's genomic material (a linear double-stranded DNA) into the bacterial cell.

In the 16th century, Galileo Galilei conducted experiments that suggested that the speed at which an object falls is independent of its mass.  This relationship, however, is strictly true only in a vacuum.

In a medium that offers resistance, such as air or water, the rate at which an object falls also depends upon its size and shape.

For bacteria and phage, the difference in sizes is dramatic.

Centrifugation assays exploit size and density differences between biological objects. In this case, we use the difference is size between bacteria and phage.

A typical E. coli is a cylinder ~1.5 µm in diameter and 2-4 µm in length (~1 to 2 cubic µm in volume); it is denser than the medium in which it grows.

Phage are even denser than bacteria but very much smaller. A T4 virion is ~0.25 µm long and 0.05 µm in diameter.

Bacteria are heavy enough to settle out of solution under the force of gravity, although it can take a few hours.

Phage are so small that the thermal motion of water molecules is sufficient to overcome the force of gravity.  Much like a feather floats on the wind, phage remain suspended in solution.

A solution of phage is a colloid - solid particles suspended in a liquid.

The speed at which bacteria "fall" can be increased by increasing the effective gravitational force -- which is exactly what a centrifuge does.

If T4 phage bind to the surface of a bacterium, a short centrifugation will remove both bacteria and phage from solution.

If the phage do not bind to the bacteria, such a centrifugation will "pellet" the bacteria, but leave the phage in the supernatant.

We will use a standard plaque assay (with wild type E. coli) to determine whether or not phage have been left in solution or removed.

To test whether our T4-resistant E. coli can still bind T4 phage, we will use a centrifugation assay.

Prepare overnight cultures of wild type and T4R E. coli strains and then add ~10⁵ phage to 1 ml of bacterial culture.

Wait 5 minutes for binding to be complete and then centrifuge the solution at 10,000g for 3 minutes.

Take the top 0.5 ml of supernatant, to avoid disturbing the pellet and determine the titer (concentration) of the phage remaining in solution.

• Check at least 3 phage resistant strains.  Which, if any, of your T4-resistant bacteria were defective in binding to wild type T4?
• Based on your previous observations, will T4 resistant bacteria that fail to bind T4 phage also fail to bind T1 phage?
• What would be the molecular reasoning behind your conclusion?
• Propose a hypothesis by which an E. coli strain could still bind T4 and yet still be resistant to infection.