Viral replication factories/site(s) inside live host: Replication forks visualized in T4infected Escherichia coli
Srivathsa Nallanchakravarthula
Every living organism maintains its continuity by passing more or less accurate copies of
its hereditary information to the next generation with the help of replication process. The
replication process can be explained as “the process by which the genetic material of an
organism copies itself in order to make a new genome to pass onto its daughter cell”.
Recent scientific studies in bacteria have shown that the replicating chromosome (genetic
material) moves through an anchored multiprotein structure, designated as a “replication
factory”.
As part of my degree project I attempted to locate and characterize the positions in
the cell where the replication factory set up for replication of the T4 bacteriophage (a
virus which infects bacteria) genomewere located. To visualize the phage development
the DNA-specific stain DAPI (4’, 6-diamidino-2-phenylindole) was used to visualize the
DNA and single strand DNA binding (SSB) protein (which binds to single stranded DNA
regions at the site of replication fork) was fused to green fluorescent protein (GFP) and
used to locate replication forks.
By examining the periodic samples of the T4 infected culture, the development of
the phage was followed using spectrophotometry (growth and lysis) and fluorescence
microscope (total DNA and replication forks in individual cells). To measure the relative
amounts of bacterial and phage DNA, flow cytometry (DNA content in cells), southern
blotting (degrading host DNA) and TCA precipitation (relative amounts of bacterial or
phage origin) were used.
The results showed (a) T4-specific foci corresponding to the early replication mode
appeared within 3-5 minutes following infection; (b) The early replication centers were
located in cytoplasmic areas free from host DNA; (c) About 10-15 minutes after
infection, the GFP spots changed from distinct to more fuzzy, indicating that late
replication (with more replicative forks per chromosome) had take over from the early
replication; (d) Phage DNA molecules formed clusters near the bacterial membrane for
packaging and lysis.
To my knowledge, this is the first time that phage infection and its development
could be observed live in real time. As expected, this novel approach raised many
interesting questions that need to be investigated further. A detailed knowledge of phage
replication and development might provide insights that would be useful in designing
approaches to phage therapy (the therapeutic use of lytic bacteriophages to treat
pathogenic bacterial infections).
Degree project in biology, Uppsala University, spring 2006
Examensarbete i biologi, 20p,
Department of Biology Education and Department of Cell and Molecular Biology,
Supervisior: Dr. Santanu Dasgupta