PHAGE MULTIPLICATION CYCLE

A. Lytic or Virulent Phages 1. Definition - Lytic or virulent phages are phages which can only multiply on bacteria and kill the cell by lysis at the end of the life cycle. 2. Life cycle . a. Eclipse period - During the eclipse phase, no infectious phage particles can be found either inside or outside the bacterial cell. The phage nucleic acid takes over the host biosynthetic machinery and phage specified m-RNA's and proteins are made. There is an orderly expression of phage directed macromolecular synthesis, just as one sees in animal virus infections. Early m-RNA's code for early proteins which are needed for phage DNA synthesis and for shutting off host DNA, RNA and protein biosynthesis. In some cases the early proteins actually degrade the host chromosome. After phage DNA is made late m-RNA's and late proteins are made. The late proteins are the structural proteins that comprise the phage as well as the proteins needed for lysis of the bacterial cell. b. Intracellular Accumulation Phase - In this phase the nucleic acid and structural proteins that have been made are assembled and infectious phage particles accumulate within the cell. c. Lysis and Release Phase - After a while the bacteria begin to lyse due to the accumulation of the phage lysis protein and intracellular phage are released into the medium. The number of particles released per infected bacteria may be as high as 1000. 3. Assay for Lytic Phage a. Plaque assay - Lytic phage are enumerated by a plaque assay. A plaque is a clear area which results from the lysis of bacteria . Each plaque arises from a single infectious phage. The infectious particle that gives rise to a plaque is called a pfu (plaque forming unit).Lysogenic or Temperate Phage 1. Definition - Lysogenic or temperate phages are those that can either multiply via the lytic cycle or enter a quiescent state in the cell. In this quiescent state most of the phage genes are not transcribed; the phage genome exists in a repressed state. The phage DNA in this repressed state is called a prophage because it is not a phage but it has the potential to produce phage. In most cases the phage DNA actually integrates into the host chromosome and is replicated along with the host chromosome and passed on to the daughter cells. The cell harboring a prophage is not adversely affected by the presence of the prophage and the lysogenic state may persist indefinitely. The cell harboring a prophage is termed a lysogen. 2. Events Leading to Lysogeny - The Prototype Phage: Lambda a. Circularization of the phage chromosome - Lambda DNA is a double stranded linear molecule with small single stranded regions at the 5' ends. These single stranded ends are complementary (cohesive ends) so that they can base pair and produce a circular molecule. In the cell the free ends of the circle can be ligated to form a covalently closed circle. b. Site-specific recombination - A recombination event, catalyzed by a phage coded enzyme, occurs between a particular site on the circularized phage DNA and a particular site on the host chromosome. The result is the integration of the phage DNA into the host chromosome. c. Repression of the phage genome - A phage coded protein, called a repressor, is made which binds to a particular site on the phage DNA, called the operator, and shuts off transcription of most phage genes EXCEPT the repressor gene. The result is a stable repressed phage genome which is integrated into the host chromosome. Each temperate phage will only repress its own DNA and not that from other phage, so that repression is very specific (immunity to superinfection with the same phage). 3. Events Leading to Termination of Lysogeny Anytime a lysogenic bacterium is exposed to adverse conditions, the lysogenic state can be terminated. This process is called induction. Conditions which favor the termination of the lysogenic state include: desiccation, exposure to UV or ionizing radiation, exposure to mutagenic chemicals, etc. Adverse conditions lead to the production of proteases (rec A protein) which destroy the repressor protein. This in turn leads to the expression of the phage genes, reversal of the integration process and lytic multiplication. 4. Lytic vs Lysogenic Cycle The decision for lambda to enter the lytic or lysogenic cycle when it first enters a cell is determined by the concentration of the repressor and another phage protein called cro in the cell. The cro protein turns off the synthesis of the repressor and thus prevents the establishment of lysogeny. Environmental conditions that favor the production of cro will lead to the lytic cycle while those that favor the production of the repressor will favor lysogeny.