Bacteriophage Isolation FROM SEWAGE

Bacteriophages are viruses that infect bacteria.  They can be found wherever bacteria are found.  Sewage is a rich source of bacteriophages that infect enteric bacteria such as Escherichia coli.  In this experiment, a filtered sewage sample will be added to a rapidly growing culture of Escherichia coli.  If phages are in the sewage sample they will go thru many cycles of infection and lyse the Escherichia coli.

Viruses are commonly characterized according to the type of cell they infect.  The three major groups of viruses are animal viruses, plant viruses and bacterial viruses (bacteriophages or simply, phages).  All viruses are obligate intracellular parasites and are incapable of functioning or reproducing outside living cells.  Viral particles are composed of a DNA or RNA core surrounded by a protein coat (capsid).  We will deal only with bacteriophages in this lab.  Two cycles of infection (lytic and lysogenic) may be exhibited by bacteriophages.  Virulent bacteriophages (e.g., T4) multiply rapidly after infecting a host cell and destroy the cell through lysis.  Temperate bacteriophages (e.g., lambda) may lyse the host cell or lysogenize the host.  If lysogeny occurs the phages produce a protein, called a repressor that prevents replication of the phage DNA. Instead the phage DNA integrates into the host genome where it is referred to as a prophage.  When the bacterial host DNA replicates, the prophage DNA is replicated as well.  Thus, all bacterial daughter cells carry a copy of the prophage DNA and are referred to as lysogenic bacteria.  The lysogenic cells may exhibit new properties such as toxin production (e.g., scarlet fever, diphtheria or botulism).

The lytic cycle of virulent phage consists of 5 sequential stages: 

1. Adsorption – A phage attaches to specific receptor sites on the surface of the host cell.  This receptor conforms to molecules present on the phage tail.  A phage cannot attack bacteria that do not have receptor molecules that conform to the phage tail proteins.
2. Penetration – The phage tail fibers contract and the baseplate settles down on the cell surface.  The phage forms a hole in the cell wall using phage lysozyme, an enzyme, and drives the tail core through the cell wall and membrane.  The phage DNA is then injected into the bacterial cytoplasm.  The head (capsid) and tail stay outside and are referred to as the phage ghost.
3. Biosynthesis - Once inside the cell, phage DNA redirects host cell metabolism.  It subverts the cell machinery to the exclusive manufacture of nucleic acid and protein molecules needed for the assembly of hundreds of new phage.  For several minutes following infection, complete phage cannot be found; this is called the eclipse period.
4. Maturation - Phage DNA, head protein and tail proteins are synthesized separately and then assembled during this step.  The head protein is packaged with phage DNA and then the tail is attached.  The eclipse period is over when the first complete phage appears in the cell.
5. Lysis - Lysozyme coded for by the phage DNA causes the host cell to burst releasing intact phages to infect neighboring cells.  For some phage, the cycle may be completed in as quickly as 15 minutes.

Bacteriophages can be found wherever their host cells reside.  If one wishes to isolate phages that infect Escherichia coli, which is found associated with fecal material, then sewage would be a good source.  If one, instead, wanted to isolate phages that infect Bacillus subtilis then soil would be a good source etc..

Rachel Watson, M.S.
AG 5010
766-3524
Cell: 307-760-2942
rwatson@uwyo.edu