Microbial genetics

MICROBIAL GENETICS

DNA as the Genetic Material

The substance that carries the information determines the prop-erties of the organism is called genetic material. This genetic material is responsible for transferring the genetic information from parent to prog-eny. In all organisms the genetic material is DNA. However, there are some exceptions such as bacteria and numerous plant and animal vi-ruses in which the genetic material is RNA.

Since the DNA is the genetic material that carries the informa-tion, it is called the central dogma, i.e., the information contained in DNA is translated to protein structures. Different experiments were carried out to identify DNA as the genetic material.

Griffith Experiment

The concept that nucleic acids are the genetic material is based on the discovery of transformation by Fredrick Griffith in 1927, medi-cal officer in the British Ministry of health, was studying Pneumococcal infection in mice. Different strains of the BacteriumDiplococcuspneumoniae, now named as Streptococcus pneumoniae were usedfor his experiments. There are two types of bacterial strains, one viru-lent and other avirulent. The virulent strains cause pneumoniae in hu-mans and mice, however, avirulent strains do not cause pneumoniae. The virulence is related to the presence of a polysaccharide capsule of the bacterium. The capsulated virulent cells cannot be phagocytosed by the circulatory system, where as non-encapsulated avirulent strains are destroyed by the phagocytic cells.

The capsulated bacteria form smooth shiny surface colony (S) when grown on agar culture plate, whereas non encapsulated strains produce rough colonies (R). Each bacterial strain may be differentiated based on serotype. The specificity of the serotype is due to the chemi-cal structure of the polysaccharide is identified by immunological tech-niques. They are usually designated by Roman numerals. There are different types such as I, II and III of Pneumococci that have different capsular polysaccharide.

In 1920’s, in United States, type I and type II were common in causing pneumoniae. Griffith book type II and type III for his experi-ments that led him to the concept about genetic material.

The characteristics of bacteria used by Griffith for his original transformation experiment is given below.

       The virulent encapsulated smooth bacteria are effective in killing the mice, whereas if the bacteria is heat killed by heat treatment, they lose their ability to kill the mice. Griffith performed a critical experi-ment. involving an injection into mice of living RII (Avirulent) cells com-bined with heat killed IIIS (Virulent)cells. Neither type can cause death in mice when injected. Griffith also expected that the double injection would not kill the mice. However, after five days all mice receiving double injection were dead. The blood analysis of all the dead mice showed a large number of living type III S bacteria. These IIIS bacte-ria were identified to the IIIS strain from which the heat killed cell prepa-ration was made. A control was maintained, where the mice received only living avirulent II R bacteria that did not develop pneumonia and remained healthy. This experiment ruled out the possibility that the aviru- lent IIR cells had simply changed to virulent IIIS cells in the absence of the heat killed III S strain.

Some type of interaction was there between living II R and heat killed III S cells. It was concluded by Griffith that the heat killed III S bacteria somehow was responsible for converting live avirulent II R cells and into virulent IIIS cells.

Griffith suggested that some transforming principle from the dead III S cells was responsible for the conversion of II R cells into III S cells. This phenomenon is called transformation. This is called as Griffith effect or more popularly called as Bacterial transformation.

Avery, MacLeod and McCarthy experiment:

Griffith could not understand the cause of bacterial transforma-tion. Oswal T Avery, C.M.MacLeod and M.J.McCarthy identified the transforming principle in 1944. Avery and his coworkers standardized a procedure to extract active power from liquid cultures of type III S virulent cells. From the original 75 liter sample, the procedure yielded 10 to 25 mg of the active factors. The “active factor” that was a fibrous mass was analyzed for its Nitrogen/Phosphorus ratio and was formed to coincide with the ratio of DNA.

The final extracted product was also treated with the proteolytic enzymes trypsin and chymotrypsin and then with an RNA digesting enzyme, ribonuclease. Such treatments destroyed the activity of pro-teins and RNA, and the transforming activity still remained.

The final confirmation was done by treating the crude samples with DNA digesting enzyme deoxyribonuclease. Digestion with this enzyme destroyed the transforming activity. This proved that the trans-forming principle was DNA.

Hershey and Chase Experiment:

Another supporting evidence that DNA as the genetic material came from the results of experiments designed by Alfred Hershey and Martha Chase in 1952.

They used T-even Bacteriophages T2. When the Bacterioph-ages infect a bacterial cell, the following events occur.

·     Attachment of phage tail fibers to bacterial wall

·     Same components of the phage enters the bacterial ell

·     The virus uses the cellular machinery of the host for viral reproduc-tion.

·     Viral components accumulate, assembly of mature phages takes place

·     Bacterial cell lysed and many new phages released.

Hershey and Chase knew from several experiments, the inde-pendent functions of phage protein and nucleic acid in the reproduction process associated with the bacterial cell.