Reproduction of bacteriophages
- 1. Reproduction of Bacteriophages Dr. R. S. Jadhav Department of Microbiology VNBN Mahavidyalaya, Shirala Dist- Sangli (MS), India.
- 2. INTRODUCTION Bacteriophages are the viruses that infect, reproduce and lyse host (bacteria) cell. Twort and d’ Herelle in 1915 and 1917 separately discovered bacteriophages. Phages or Bacteriophages (Bacteria eating viruses) are intracellular parasites of bacteria. Bacteriophages inserted nucleic acid into host cell and reproduce using host biosynthetic machinery. The capsid remains outside the cell. In replication and expression of nucleic acid produce more copies of nucleic acid and capsid protein and assemble together to form new virus particles. we study two point in this topic Reproduction of bacteriophages such as 1) One step growth experiment/ One step multiplication curve 2) Multiplication of T4 bacteriophages.
- 3. 1) One step growth experiment/ One step multiplication curve One step growth experiment was first carried out by Emory L. Ellis and Max Delbruck in 1939 using bactreiophages. They also performed the plaque counting method for bacteriophage quantification. This study represents the started of molecular virology, molecular biology and molecular genetics. Max Delbruck were awarded the Nobel prize in 1969. In this experiment study reveals fundamental nature of virus replication. One step growth means single or one cycle of virus growth is observed . In this experiments, excess host cells are infected by phage particles, which make the infection synchronous. The observation made on a host cell culture is similar to the observation made on single cell infected by a phage. The excess host cells are infected with phage particles at a ratio of 1 to 10. the idea is to prevent adsorption of more than one virus per cell. The mixture is allowed to stand for 5 min. during this period phage adsorb on host cells. The mixture is then diluted 1:1000 in broth to eliminate immediate adsorption of virus particles released upon host cell lysis. Thus, only one step virus growth. Sample of dilute mixture are removed at regular time interval and plated for plaque count. Assay in this case gives a measure of infectious centers i.e. the number of virus particle and infected bacteria (No.of plaque /ml) and the end of burst size . When log number of plaque forming units/ml is plotted against time, a curve of the form shown in fig. 1 is obtained. The curve gives three distinct phases: 1) Latent period 2) Brust period or rise period 3) Plateau period
- 4. 1) Latent period- (Eclipse + Intracellular accumulation)- The time form infection until cell lysis is called as latent period. Thus there is no release of new viral particles from infected cells and so plaque count remains constant ( each infected cell gives only one plaque). T2 has latent period of around 22-23 min at 37 degree Celsius. The latent period can be divided into : Eclipse and Intracellular accumulation period. Eclipse period- The time from infection until intracellular accumulation of phages is called as eclipse period. T2 has about 11.5 min at 37 degree Celsius eclipse period. In this period , gene expression, protein and genome synthesis occure. Intracellular accumulation: The time for initiation to end of Intracellular accumulation of phages is called as intracellular accumulation period. Here phage proteins and genomes assemble into new phage particles. The intracellular accumulation of T2 phage requires about 11-12 min at 37 degree Celsius. Fig: 1
- 5. 2) Burst or Rise period: The time from infected host cell lysis initiation to end is called rise or burst period. At the end of latent period each infected cell lyses and liberates new virus particles. In this phase, new virus particle releases and plaque count rises rapidly (each new virus particle forms a plaque on lawn of susceptible host cells). T2 has a rise period of about 10 min at 37 degree Celsius. Due to ansynchrony of infection the rise period is slightly extended. 3) Plateau peroid: Plateau represents end of all infected host cell lysis. The newly liberated phage particle fail to meet uninfected host cells due to high dilution. Thus here the plaque count remains constant. Plateau period of T2 phage is 30 min. at 37 degree Celsius. Burst size: Burst size refers to the number of virus particles produced from the infection of a single cell. The burst size is calculated by using following formula: p.f.u/ml at plateau Burst size: ----------------------------------------- p.f.u./ml at latent period The T2 phage has a burst size of >100 phages per cell. The burst size varies from 20 to 3000virions/ cell for different viruses.