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Role of Bacteriophages in Genetics and Molecular Biology

By deepone Apr 03, 2011 2522 Words

A bacteriophage is any one of a number of viruses that infect bacteria. Bacteriophages are among the most common biological entities on Earth. The term is commonly used in its shortened form, phage.Typically; bacteriophages consist of an outer protein capsid, enclosing genetic material. The genetic material can be ssRNA, dsRNA, ssDNA, or dsDNA along with either circular or linear arrangement. Bacteriophages are much smaller than the bacteria they destroy. In retro respect, there are a few reports in the literature that hint at the presence of bacteriophages. Hankins (1896) reported that the waters of Jumna and Ganges rivers in India had antiseptic activity against many kinds of bacteria and against the cholera vibrio in particular.  In 1915, British bacteriologist Frederick Twort, superintendent of the Brown Institution of London, discovered a small agent that infected and killed bacteria. He believed that the agent must be one of the following: 1. A stage in the life cycle of the bacteria;

2. An enzyme produced by the bacteria themselves; or
3. A virus that grew on and destroyed the bacteria.

Twort's work was interrupted by the onset of World War I and shortage of funding. Independently, French-Canadian microbiologist Félix d'Hérelle, working at the Pasteur Institute in Paris, announced on September 3, 1917, that he had discovered "an invisible, antagonistic microbe of the dysentery bacillus".D'Hérelle called the virus a bacteriophage or bacteria-eater (from the Greek phagein meaning to eat). He also recorded a dramatic account of a man suffering from dysentery who was restored to good health by the bacteriophages. He found that the antigenic properties and host-range specificity of phages appeared to be characteristic of given “races” of phages. Thus from the beginning, there were hints that phages might be fruitful organisms for genetic study. Duckworth (1976) provides a detailed historical account of the discovery of bacteriophages. ROLE OF PHAGES:

The early history of bacteriophages might suggest that their study would immediately be applied to the treatment of diseases caused by bacteria. This was not the case; instead antibiotic therapy became the mainstay of treatment for bacterial diseases. However, the importance of the bacteriophage in the advancement of biological science cannot be overstated. Bacteriophages were intensively studied in the decades after their discovery, and came to play a leading role in the advancement of the basic science of microbiology and the new biology of molecular genetics. In the 1940's, and onward, it was the laboratory study of phage biology that directly yielded major insights into bacterial genetics, molecular biology, and the exact manner in which viruses reproduce and spread. These discoveries include:

1. Mutations arise in the absence of selection (Luria and Delbruck 1943).

The Luria–Delbruck experiment (1943) (also called the Fluctuation Test) demonstrates that in bacteria, genetic mutations arise in the absence of selection, rather than being a response to selection. In their experiment, Luria and Delbruck inoculated a small number of bacteria into separate culture tubes. After a period of growth, they plated equal volumes of these separate cultures onto agar containing phage (virus). If virus resistance in bacteria were caused by a spontaneous activation in bacteria—i.e., if resistance were not due to heritable genetic components, then each plate should contain roughly the same number of resistant colonies. This however was not what Delbruck and Luria found. Instead, the number of resistant colonies on each plate varied drastically. Luria and Delbruck proposed that these results could be explained by the occurrence of a constant rate of random mutations in each generation of bacteria growing in the initial culture tubes. Based on these assumptions Delbruck derived a probability distribution (now called the Luria–Delbruck distribution) that gives a relationship between moments consistent with the experimentally obtained values. The distribution that follows from the directed adaptation hypothesis (a Poisson distribution) predicted moments inconsistent with the data. Therefore, the conclusion was that mutations in bacteria, as in other organisms, are random rather than directed.

2. Genetic transduction (Zinder and Lederberg 1952).

Phage mediated transduction of a small segment from any region of the bacterial chromosome is called generalized genetic transduction. In 1952 Zinder and Lederberg discovered that bacteriophage particles could transfer bacterial genes from one bacterium to another. Zinder and Lederberg initially began their experiments with the objective of discovering whether the E. coli type of genetic exchange also existed in Salmonella typhimurium. Various strains of auxotrophs were crossed on an amino acid free minimal medium in an attempt to find new prototrophic combinations. It is only occasionally that a single phage will transduce several gene loci (multiple transductions). Linkage maps for short sequences of genes can be constructed by using multiple transductions. Generalized transduction resembles transformation in the respect that usually only a single marker locus is transduced. The difference between the two processes is that in transduction the DNA reaches the cell surrounded by the phage coat, while in transformation the DNA is naked. Transduction, therefore, has an advantage in mapping because it protects the DNA from the environment, and thus makes it more readily reproducible.

3. DNA is genetic material (Hershey and Chase 1952).

The Hershey–Chase experiments were a series of experiments conducted in 1952 by Alfred Hershey and Martha Chase, confirming that DNA was the genetic material. Hershey and Chase conducted their experiments on the T2 phage, a virus whose structure had recently been shown by electron microscopy. The phage consists of a protein shell containing its genetic material. The phage infects a bacterium by attaching to its outer membrane and injecting its genetic material and leaving its empty shell attached to the bacterium. In their first set of experiments, Hershey and Chase labeled the DNA of phages with radioactivePhosphorus-32 (the element phosphorus is present in DNA but not present in any of the 20 amino acids from which proteins are made). They allowed the phages to infect E. coli, and through several elegant experiments were able to observe the transfer of P32 labeled phage DNA into the cytoplasm of the bacterium. In their second set of experiments, they labeled the phages with radioactive Sulfur-35. Following infection of E.coli then sheared the viral protein shells off of infected cells using a high-speed blender and separated the cells and viral coats by using a centrifuge. After separation, the radioactive S35 tracer was observed in the protein shells, but not in the infected bacteria, supporting the hypothesis that the genetic material which infects the bacteria was DNA and not protein. Hershey shared the 1969 Nobel Prize in Physiology or Medicine with Chase for their “discoveries concerning the genetic structure of viruses.”

4. Restriction and modification (Luria and Human 1952; Dussoix and Arber 1962).

The phenomenon of restriction and modification was first observed genetically in 1952 [Luria and Human, 1952] and 1953 [Bertani and Weigle, 1953], although they referred to it as host induced or host controlled variation. Restriction and modification of phages by bacteria were first described more than 40 years ago by Luria and Human (1952) for T-even phages and by Bertani and Weigle (1953) for the phages P2 and λ. The basic observation was that phages grown on one bacterial strain would grow poorly when tested on certain other related bacterial strains. Upon closer investigation, the poor growth was found to be an all or nothing affair. Most infected cells produced no phage at all; in the terminology that was adopted, the phages were restricted, although a few cells produced a normal yield of phage particles. The few phages that did result from the infection were modified so that they grew normally on the new host when tested in a second cycle of infection. Often, they were now restricted by the original host. The phenomenon of modification is Lamarckian rather than Mendelian in character since it is an adaptive response of the virus to the host that is lost when the virus is pass aged through other cells. Some 10 years after the discovery of restriction and modification, the first of a series of papers from Arber’s laboratory appeared that offered a molecular explanation for the effect (Arber and Dussoix 1962; Dussoix and Arber 1962). Both restriction and modification were properties of the DNA of the infecting phage. A significant breakthrough came in 1970 when the first type II restriction enzyme was reported that was able to cleave bacteriophage T7 DNA into specific fragments.

5. Genetic fine structure (Benzer 1955).

Benzer developed the T4 rII system, a new genetic technique involving recombination in mutant bacteriophage. Taking advantage of the enormous number of recombinants that could be analyzed in the rII mutant system, Benzer provided the first evidence that the gene is not an indivisible entity, as previously believed. Benzer proved that mutations were distributed in many different parts of a single gene, and the resolving power of his system allowed him to discern mutants that differ at the level of a single nucleotide. Benzer's experiments with the rII system are widely considered among the most elegant experiments in modern genetics. In his molecular biology period, Benzer dissected the fine structure of a single gene, laying down the ground work for decades of mutation analysis and genetic engineering.

6. Messenger RNA (Volkin and Astrachan 1956).

Volkin infected bacterial cells of Escherichia coli with the bacteriophage virus, added phosphorus-32, isolated nucleic acid from the preparation, and hydrolyzed it with sodium hydroxide to make alkaline products that were separated using ion-exchange chromatography. The results of experiments with phosphorus-32 were confirmed using a carbon-14 precursor that was specifically incorporated into the nucleic acid bases. Larry Astrachan joined Volkin in performing these experiments, which led to the discovery of messenger RNA, but they called it "DNA-like RNA." According to Berg, the ORNL researchers "discovered that the virus 'turns off' the [bacterial] cell's machinery for making its own proteins and 'instructs' the cell's machinery to make proteins characteristic of the virus. That instruction entails making a new kind of RNA, a copy of the virus's DNA. This discovery revealed a fundamental mechanism for gene action: the coding sequences of genes are copied into short-lived RNAs that are transported out of the nucleus into the cytoplasm, where they are translated into proteins. Because such RNAs transport information from genes in the nucleus to the cytoplasm, they are designated as messenger RNAs."

7. Acquisition and loss of genes from genomes (Campbell 1962).

Allan Campbell proposed in 1962 that λ attaches to the bacterial chromosome by a reciprocal crossover between the circular λ chromosome and the circular E. coli chromosome. Campbell’s model for the integration of phage λ into the E. coli chromosome. Reciprocal recombination takes place between a specific attachment site on the circular λ DNA and a specific region on the bacterial chromosome between the gal and bio genes.

8. Molecular basis of DNA recombination (Meselson and Weigle 1961).

Meselson and Weigle infected E. coli cells at the same time with phage from two different stocks of bacteriophage lambda. One stock had been prepared by growing the bacteriophage lambda c-mi- in cells grown in medium containing heavy isotopes of carbon (13C) and nitrogen (15N). The other stock had been prepared by growing bacteriophage lambda c+mi+ in medium containing light isotopes of carbon and nitrogen. After infection, the progeny phage were isolated and banded on a CsCl gradient. A broad band of phage particles were found on the gradient.

* Non recombinant phages were found, as expected, at two well-defined densities corresponding to the parental light and heavy phages.  * Recombinant phages were found - surprisingly - at all intermediate densities between these two. These results can only be explained if recombination between the two parental phage involves breakage and rejoining of both DNA strands.

9. Gene regulation (Jacob and Monod 1961).

Jacob and Monod used phages in their experiments to study and explain gene regulation. The regulation of gene activity has developed into a very large sub-discipline of molecular biology, and in truth exhibits enormous variety in mechanism and many levels of complexity. Current researchers find regulatory events at every conceivable level of the processes that express genetic information.

10. Triplet nature of DNA code (Crick, Barnett, Brenner and Watts-Tobin 1961).

In the late 1950s an assay using phage mutations which provided the first detailed linearly structured map of a genetic region, was made. Crick felt he could use mutagenesis and genetic recombination phage to further delineate the nature of the genetic code. In the experiment, using these phages, the triplet nature of the genetic code was confirmed. They used frameshift mutations and a process called reversions, to add and delete various numbers of nucleotides. When a nucleotide triplet was added or deleted to the DNA sequence the encoded protein was minimally affected. Thus, they concluded that the genetic code is a triplet code because it did not cause a frameshift in the reading frame.


11. DNA and protein are collinear (Sarabhai, Stretton, Brenner and Bolle 19640. 12. DNA ligase (Gellert 1967)
13. DNA recognition and cooperative binding (Ptashne 1967) 14. chaperones and protein folding (Georgeopoulos, Hendrix, Casjens and Kaiser 1973) 15. first DNA genome sequenced, phiX174 (Sanger et al. 1977) 16. epigenetic gene regulation (Ptashne 2004)

17. repression and activation (i.e. turning genes on and off)

* Food safety:
In August, 2006 the United States Food and Drug Administration (FDA) approved using bacteriophages on certain ready-to-eat meats to kill the potentially lethal Listeria monocytogenes bacteria. The additive, known as LMP-102™, is a proprietary cocktail of six bacteriophages specific for Listeria, from Intralytix, Inc. Intralytix is also seeking FDA approval for Escherichia coliO157:H7 and Salmonella treatments.

* Nanotechnology:
Another large use of bacteriophages is by the company Cambrios Technologies. Its founder, Dr Angela Belcher, pioneered the use of the M13 bacteriophage to create nanowires and electrodes. She started her research by studying how abalone snails create their shells from things that naturally occur in their environment. Specifically, she discovered the snails take abalone and make them transform into two distinct crystalline structures. One of the structures was hard; the other was fast-growing. She took this concept and applied it to bacteriophages. One of her ventures consisted of implanting gold and cobalt oxide in a bacteriophage to create a paper-thin electrode: the gold was for conductivity, and the cobalt oxide was for the actual use of the battery.

* Bacteriophage experimental evolution:
Experimental evolution is the use of model organisms (e.g. coli, yeast, lambda phage) to study the process of evolution in controlled experiments. Because of their rapid generation times, small sizes, ease of manipulation, small genomes and availability of molecular genetic and biochemical details, bacteriophages are ideal organisms for experimental studies of evolution. Phage experimental evolution is part of the broader field of viral evolution.

* Phage therapy.

* *

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