Effects of Bacteriophages on Modern Biology

By Kristi McKee

Scientific Discovery

Fall 2003

What is a Bacteriophage

Viruses that attack bacteria Non-self replicating Made up of mostly proteins and Deoxyribonucleic

acids (DNA) Bacteria specific Inject their genome into host cell

– Lytic cycle– Lysogenic cycle

Phage Appearance

Average length is around one twenty-fifth of a micron, but vary in size.

First use of how phages can manipulate the cells production was producing human insulin.

What phages do to Host Cell

www.cellsalive.com

Example of Lytic Life Cycle

http://www.cat.cc.md.us/courses/bio141/lecguide/unit2/viruses/rellyso.html

Discovery of Bacteria Infecting Viruses

Frederick W. Twort given first credit for phages.

Found by studying micrococcus colonies

Naming of the “Viruses”

Felix D’ Herelle

Born in Montreal 1873– Trained in medical

bacteriology

Came across first in Mexico with locusts

What D’Herelle Did and Found

Collected sick locusts and studied the blackish diarrhea.

In all samples found bacteria locust coccobacillus

Took healthy colonies and dusted plants that the insects ate and watched them as well become infected.

He tested this dust all over the world.

What D’Herelle Did and Found

Also set up agar plates with colonies of coccobacilli that was collected from insects.

Made a slide from the agar but saw nothing under the microscope

Decided that the cause was a filterable. So he used a porcelain filter that would retain all bacteria.

The Dawning of the Light

1915 visiting Paris asked to investigate an epidemic of dysentery in the Calvary squadron.

Decided to follow one patient from the time they were admitted till end of convalescence to see at what time the principle causing agent of the clear spots occurred.

Results of Tracking Patient

1st day normal growth on plate 2nd and 3rd no changes 4th day made emulsion with a few drops from

the bloody stools, filtered through a Chamberlain candle, added to broth culture of dysentery bacillus isolated from 1st day. Filtrated again and spread to a plate and broth culture. Incubated at 37o.

The Morning After

The broth culture that was turbid, cleared as well as the agar spread was also devoid of all growth.

Went to hospital to share his results and check on patient.

Reaction of D’Herelle

"...on opening the incubator I experienced one of those rare moments of intense emotion which reward the research worker for all his pains: at first glance I saw that the broth culture, which the night before had been very turbid was perfectly clear: all the bacteria had vanished... as for my agar spread it was devoid of all growth and what caused my emotion was that in a flash I understood: what causes my spots was in fact an invisible microbe, a filterable virus, but a virus parasitic on bacteria. Another thought came to me also, If this is true, the same thing will have probably occurred in the sick man. In his intestine, as in my test-tube, the dysentery bacilli will have dissolved away under the action of their parasite. He should now be cured"

The Light Comes On

From all the results D’Herelle now had he knew it was a filterable virus that was parasitic on bacteria.

He coined the name bacteriophages Tried to use phages to treat and prevent

other diseases such as: anthrax, typhus and bubonic plague.

Short Life of Phage Therapy

After 1920’s there was a skyrocket in the use of phage therapy.

Even as late as WWII phage therapy was being used prevalently in some countries.

However, antibiotics began to phase them out.

Beginnings of Phage Research

FM Burnet – origins of phage research Showed that bacteriophages vary widely in

physical and physiological properties Different phages carry different antigens Proposed single-burst method

Discovering Linear Dimensions

Martin Schlesinger – studied properties of phages by their chemical and physiochemistry methods to give first insight of what a phage may look like.

Showed they are ~0.1m in length and ~4x10-10g.

Found that phages consisted of mainly proteins and DNA in equal proportions.

Max Delbruck

He is often referred to as the founder of molecular biology.

Perfected a method to culture phages and found they can infect a cell and with in thirty minutes lyse cell and have hundredfold their original number – one step growth method

Along with Hershey found bacteria can spontaneously mutate to become immune to phages.

Delbruck continued

Worked with many other scientists to find that viruses contain DNA.

As well as helped to reveal the structure of DNA itself.

Awarded the Nobel for work on phages in 1969 along with Alfred Hershey and Salvador Luria.

Found that phages could also after infecting a cell phages could exchange genetic information to form new viral strains

First Electron Micrograph

Luria and Anderson – 1942 first electron micrograph picture of a T2 phage

Anderson also discovered the phages adsorbed by the tail by “critical point” technique for avoiding surface tension.

Theoretical goes Experimental

Through the theoretical contributions of Delbruck and Luria, Hershey used their findings to produce his skillful experimentation along with Martha Chase.

Penetration of Virus to Host Cell

“Blender Experiment” – 1952 Alfred Hershey and Martha Chase.

Wanted to demonstrate that DNA not protein was the genetic material of life.

Early Evidence that DNA is the Substance of Life

Miescher – isolated DNA from salmon sperm found DNA is made up of 4 monomers.

First half of 20th century nucleic acids were thought to be only structural material in the cells nucleus.

Hershey and Chase’s experiment was benchmark in changing ideas.

Focus of Study

Study involved T2 bacteriophages that infected Escherichia Coli.

The “Blender Experiment”

Wanted to show that the DNA passed on was the signal for replication and growth

1951 – 52 using radioactive phosphorus and sulfur they grew phages on media.

Then let phages infect a bacteria culture to replicate. The process was interrupted by spinning the culture

in an everyday Waring kitchen blender. The shearing of the blender separated phage protein

from bacteria cells.

“Blender Experiment” cont.

The cultures were spun in a centrifuge to separate the liquid from the bacteria that formed a pellet at the bottom.

Using the tags as a marker for where the phage protein and DNA was gave the results and proof they had been hoping for.

DNA, not protein, was the genetic material of life.

www.accessexcellemce.org

Leads for Future Experiments

Scientist now wanted to know how DNA: Operated Replicated Direct the production of proteins What was its structure

Results from Learning of Phages

1970 Hamilton Smith found first site-specific restriction enzyme using phages.

Antibiotic resistant bacteria: use of phages to destroy bacteria.

Search for new potential drugs. Develop human antibodies. Biosensors Only ~0.0002% of global phage genome has been

sampled.

Resources

“Alfred Day Hershey”, Discovering Science Bibliography “Max Delbruck”, Discovering Science Bibliography “T4 Bacteriophages attacking an E. Coli Bacterium”,

Discovering Science Evers, Chris. “Experiments that Inspire”

www.acessexcellence.org Mathews, Christopher K.; VanHolde, KE.; Biochemistry

2nd Ed. Benjamin/Cummings Publishing Company, Inc, Menlo Park Ca. 1996. pp 93, 94.

“Max Delbruck – Nobel Lecture” www.nobel.se

Recourses Cont.

“The Nobel Prize in Physiology or Medicine 1969” www.nobel.se

Rohwer, F. 2003 “Global phage diversity”. Cell 113 (April 18) :141.

Seaman, Tracy and Trollip, Andre and Mole, Richard and Albert, Heidi (2003) “The use of a novel phage-based technology as a practical tool for the diagnosis of tuberculosis in Africa”. African Journal of Biotechnology 2(2):40-45.

Travis, John. “All the World’s a Phage” Science News (2003) Vol. 164(2)