The following points highlight the top six uses of mutations in microorganisms. The uses are: 1. Determination of Function 2. Demonstration of Metabolic Pathways in Microorganisms 3. For Understanding The Metabolic Regulation 4. For Matching a Biochemical Entity with a Biological Function 5. For Locating the Site of Action of External Agents 6. For the Production of Useful Products.
Use # 1. Determination of Function:
A mutation defines a function. For example, a wild type E.coli cells can uptake lactose from 10-5 M solution by a passive diffusion through the cell membrane. But the mutants cannot uptake lactose even at the concentration higher than 10-5 M. This shows that the genetically determined process is involved in lactose uptake.
Use # 2. Demonstration of Metabolic Pathways in Microorganisms:
It has been demonstrated by isolating the three different classes of gal mutants that galactose is utilized by three distinct genes, galK, galT and galE. The Gal+ cells are grown on medium containing radioactive galactose (14C-Gal). As the 14C-Gal is metabolized, many different radioactive compounds can be found in the growth medium.
In the beginning of addition of 14C-Gal three radiolabelled compounds viz., 14C-galactose 1-phosphate (Gal-1-P), 14C-uridine di-phospho-galactose (UDP-Glu) and 14C-uridine di-phosphoglucose (UDP-Gal) are detected. If mutation occurs in three different genes, it will block the specific step of metabolic pathway.
For example, the mutant cells containing a galK– mutation would not be able to metabolize 14C- Gal, therefore 14C galactose remains unutilized because galK gene product converts into the first metabolic product. The galT-mutant cannot convert the first metabolic product into the second product, but accumulates Gal-1-P.
This shows that the first step of galactose metabolism is the conversion of galactose by galK gene product to gal-1-P. However, if galE mutant is used the UDP- Gal is found in the culture medium. This shows that galE gene is associated with conversion of UDP-Gal into a product X.
On the basis of conversion of these products the biochemical pathway must be as below:
Use # 3. For Understanding The Metabolic Regulation:
Several bacterial mutants have been isolated which show changes in amount of a particular protein of its responses to external signals. For example, the enzyme synthesized by galK, galT and galE genes are normally not present in bacteria. These are synthesized only when galactose is supplied in the growth medium.
In addition, such mutants have also been isolated in which these enzymes are always present irrespective of presence or absence or galactose in the growth medium. This shows that a regulatory gene must be associated for switching on or switching off the enzyme production.
Use # 4. For Matching a Biochemical Entity with a Biological Function:
E. coli synthesizes an enzyme, DNA polymerase which polymerizes the DNA. It was thought that DNA polymerase I is also synthesizes the bacterial DNA. In Pol A-mutant of E.coli the activity of polymerase I has been found reduced by 50 time. After biochemical analysis of cell extracts of Pol A- mutants of E.coli two other enzymes, DNA polymerase II and DNA polymerase III were isolated. The purified enzymes synthesized the DNA molecule.
In another study, a temperature sensitive (Ts) mutation in dnaE gene was detected which was not able to synthesize DNA at 42°C, but synthesized the DNA normally at 30°C. From the culture of DnaE– (Ts) mutant the enzyme DNA polymerase I, II and III were isolated and assayed separately. It was found that DNA polymerase I, II and III were active at 30°C and 42°C, and polymerase III was active only at 30°C but not 42°C.
Use # 5. For Locating the Site of Action of External Agents:
An antibiotic, rifamycin is known to inhibit RNA synthesis. In the beginning it was unknown about the precise activity of rifamycin whether it acts by checking the synthesis of precursor molecule by binding to DNA and in turn by inhibiting the transcription of DNA into RNA, or by binding to RNA polymerase.
Two types of rifamycin resistant mutants were isolated. First with altered cell wall in which rifamycin could not enter, and the second with altered RNA polymerase. These findings prove that the antibiotic rifamycin acts by binding with the enzyme RNA polymerase.
Use # 6. For the Production of Useful Products:
In addition, mutation in microorganisms for beneficial products was started since the time of Alexander Fleming during the end of 1920s. By using ultraviolet rays penicillin production by a mold Pencillium chrysogenum has been increased by about thousand fold greater to what was produced at Fleming’s time.