EXERCISE 6

SELECTION OF BACTERIOPHAGE RESISTANT MUTANTS

I. INTRODUCTION

Bacteria have their own viruses that attack them and replicate with the assistance of the bacterial biosynthetic machinery. These bacterial viruses, or bacteriophage, attach to the bacterial cell surface. Once attached, the nucleic acid or genome of the bacteriophage enters the bacterial cell and begins to express all of the viral genes necessary for the biosynthesis, assembly, and release of the progeny bacteriophage.

Bacteria can become "immune" to infection by a specific bacteriophage when they lose the ability to express the specific bacteriophage receptor on their cell surface. Without the receptor, the bacteriophage cannot attach to the bacterium and infection cannot take place. Bacteria lose the ability to express the cell surface protein by mutation of their DNA. Mutant bacteria resistant to bacteriophage infection may be isolated from a population of susceptible bacteria because the bacteriophage will cause the death of all susceptible bacteria (leaving only the bacteriophage resistant bacteria alive). The bacteriophage in this case acts as an indicator of resistance; it does not cause the resistance to occur.

Generally, genes mutate spontaneously at a frequency of 1 in a million to 1 in a billion per cell per generation. This means that out of a population of 1 billion bacteria (about 1 ml of a stationary phase culture), there is likely to be from 1 to 1000 bacteria that have the mutation in which you are interested. This laboratory exercise shows the frequency by which mutations arise in bacteria and the ease with which some mutants can be isolated. The bacteriophage you will use is called "sewer-phage" 108. It specifically attaches to a major outer membrane porin protein of E. coli called the Omp-F protein. It is through this Omp-F porin that penicillin passes into the bacterium.

II. LAB WORK

• 1 tube of ESCHERICHIA COLI (red cap)
• 1 tube of BACTERIOPHAGE SŲ108 (yellow cap)
• 1 Nutrient Agar plate (NA)
• 1 sterile Pasteur pipet
• 1 sterile cotton swab

1. Label the Nutrient Agar plate with your name.
2. Resuspend the broth culture of E. coli by mixing.
3. Moisten a cotton swab in the broth culture (not the lysate!) and spread the sample over the entire surface of the plate (as if you were painting the whole area). The surface of the agar should be swabbed by first "painting" in one direction, rotating the plate 90 degrees, and "painting" again. Do not re-wet the swab. Discard this swab correctly.
4. When the surface of the agar is DRY, after 3-4 minutes, use the sterile Pasteur pipet and rubber bulb (in drawer) to place one drop of the bacteriophage solution in the center of the plate. If the plate surface is wet, the lysate drop will spread excessively. Discard this pipet properly.
5. Allow the drop to soak into the agar a few minutes before you place the plate in the 37C incubator. DO NOT INVERT THESE PLATES.

   NEXT LAB PERIOD

6. Observe the plate and describe what you see. Return the plate to the 37C incubator until the next lab period.

   THIRD LAB PERIOD

7. Several days have elapsed since you originally spotted your plate with bacteriophage. Observe the plate and record your results. Look very carefully; are the results different from before?

Exercise 6 Results

Is the mutation caused by the bacteriophage? What would you predict the penicillin sensitivity of these bacteria to be? How would you prove it?