Genetic transformation in Prokaryotes

Genetic transformation in prokaryotes has led to the discovery of the three major methods of transformationin bacteria i.e transformation, conjugation and transuction whichcommonly uses the bacterial- phages as vectors to transfer dna.

1. DAVANGERE UNIVERSITY SHIVAGANGOTHRI – 577 007 Department of Studies in Microbiology. Seminar Topic On : GENETIC TRANSFORMATIONS IN PROKARYOTES Presented By: Dennis M. Mondah MSc 2 nd Semester Dept. of Microbiology. Under the guidance of: Dr. Vinay Kumar P.G Dept. of Microbiology Presented On: 29 th June 2021.

2. CONTENTS: Introduction Types of genetic Transformation in prokaryotes 1.Transformation 2. Conjugation 3.Transduction Transformation - Griffiths experiment - Mechanism of transformation - Methods of Transformation Conjugation - Mechanism of Conjugation Transduction - Types of transduction - Lytic & Lysogenic Cycles Applications of Genetic transformation in Prokaryotes Summary Conclusion References

3. Introduction Genetic transformation in prokaryotes is the process through which microorganisms mostly bacteria and archaea acquire foreign DNA material fragments from their surrounding to bring about genetic variation and diversity. Prokaryotes are unicellular microorganisms which have DNA as a genetic material which exists as a single, circular chromosome. Reproduction in prokaryotes is through asexual method which is mostly by binary fission where the bacterial cell enlarge and divide along the equator and the resulting cells are similar genetically. For genetic variations to occur in prokaryotes the are some mechanisms for gene transfer to occur which increases their gene diversity.

4. Types of prokaryotic Genetic transformation The most commonly methods of gene transfer in prokaryotes are; Transformation Transduction Conjugation Transformation- This is the transfer DNA where the bacterial cell takes in DNA fragment from the environment which has been shed by a dead lysing bacteria into the surrounding. Transduction- This the transfer of DNA from one cell to another by the virus mostly the bacteriophage which infect the bacteria and take control of its replication machinery forming new virus particles. Conjugation- This is the transfer of DNA directly from one cell to another through cell-cell contact using conjugation tubes. The DNA transferred involves plasmids which can replicate in the bacterial cell independently of the chromosome.

5. 1. Transformation In this method of DNA transfer the bacterial cell takes up the naked DNA from its surrounding environment and incorporates it to its genome. Transformation was first studied by Fredrick Griffith a British bacteriologist in 1928   and found out that one form of the pathogenic pneumococci (Streptococcus pneumoniae) could be “transformed ” into another form. •  Griffith showed that virulence factors from a killed pathogenic strain could “transform” a non-virulent strain to become virulent as well. The theory of transformation was upheld by Oswald Avery, Maclyn McCarty and Colin Macleod In 1944 and concluded that DNA is responsible for genetic transformation. Transformation using electroporation was developed in 1980s.

6. Fredrick Griffith Experiment. In his experiments, Griffith used two related strains of Streptococcus pneumoniae bacteria known as R and S- strains. R strain.  When grown in a petri dish, the R bacteria formed colonies, or clumps of related bacteria, that had well-defined edges and a rough appearance (" R"). The R bacteria were non-virulent , meaning that they did not cause sickness when injected into a mouse. S strain.  S bacteria formed colonies that were rounded and smooth ("S "). The smooth appearance was due to a polysaccharide, or sugar-based, coat produced by the bacteria. This coat protected the S bacteria from the mouse immune system, making them virulent. Griffith injected both S and R strains to mice. The one which was infected with the S strain developed pneumonia and died while that infected with the R strain stayed alive. In the second stage, Griffith heat-killed the S strain bacteria and injected into mice, but the mice stayed alive. Then , he mixed the heat-killed S and live R strains. This mixture was injected into mice and they died. In addition, he found living S strain bacteria in dead mice . Based on the observation, he concluded that R strain bacteria had been transformed by S strain bacteria. The R strain inherited some ‘transforming principle’ from the heat-killed S strain bacteria which made them virulent.

7. Fredrick Griffith Experiment.

8. Mechanism of Transformation. Bacterial competence- This refers to the state of the microorganism to be able to take up the DNA. Competence is of two types: 1. Natural competence 2. Artificial competence Natural Competence- This is where some bacterial species are naturally capable to take up DNA since the posses some mechanism or sets of genes which can transport DNA across the cell membrane. Artificial Competence- this refers to the laboratory manipulation of bacterial cells to make them passively permeable to the exogenous DNA. Competence can be increased by physical or chemical treatment. Competent bacterial cell can intake rDNAs with the size less than 15 kbp from the culture. The competence may be transient or few minutes in some sp., (streptococcus) while in others it can last for more than an hour(bacillus).

9. Molecular mechanism of Transformation In 1974 Notani and Setlow described the mechanism of bacterial transformation. In S. pneumoniae the competent state is transient and persists only for a short period. The competent state is induced by the competence activator protein that binds to the plasma membrane of receptor and triggers the synthesis of 10 new proteins within 10 minutes .   The competence factor (CF) accelerates the process of transport or leakage of autolysin molecules into the periplasmic space. Structural changes in competent cells induce numerous vesicles called transformosome buds on the surface that contains protein and facilitates the uptake of transforming DNA

10. Process Of Transformation The process of genetic transformation in bacteria occurs in the following steps; DNA binding DNA penetration Synapsis formation Integration Some factors that facilitate transformation are: Molecular size and weight of DNA ( 3 -8 million) DNA concentration in the medium Growth Phase of the bacterial cells.

11. A). DNA binding As a result of random collision, DNA comes first in the contact of cell surface of competent bacteria . First the DNA binding is reversible and takes for about 4-5 seconds. Thereafter , it becomes irreversible permanently. For about 2 minutes it remains in non-transforming state. Thereafter , before 5 minutes it is converted into the transforming state. The period (about 10 minutes) during which no transformation occurs in competent recipient cells is called eclipse. Both types of DNA, transforming and non-transforming, bind to the cell surface where the receptor sites are located. In H. influenza transformosome bud forms the surface and contains proteins that mediate DNA uptake . In S. pneumoniae the CF induces the ability to bind DNA molecules.

12. B). DNA Penetration- The DNA molecules that bind permanently and enter the recipient cells. DNA is also resistant to DNase degradation. The nucleolytic enzymes located at the surface of competent recipient cells act upon the donor DNA molecule when it binds to the cell membrane. The endonuclease-1 of the recipient cells which is associated with cell membrane acts as DNA translocase by attacking and degrading one strand of the dsDNA. Consequently only complementary single strand of DNA enters into the recipient cells . (It has been confirmed by performing the experiments with radiolabelling of donor DNA. E.g. In B. subtilis degradation of one strand is being delayed. Hence, both the strands enter the recipient cell). Successful transformation occurs with the donor DNA of molecular weight between 30,00,000 and 8 million Dalton. With increasing the concentration of donor DNA the number of competent cells increases. After penetration the donor DNA migrates from periphery of cell to the bacterial DNA. This movement in different bacteria differs.

13. C). Synapsis Formation- The single stranded DNA is coated with SSB proteins, which maintain, the single stranded region in a replication fork . The single strand of the donor DNA or portion of it is linearly inserted into the recipient DNA. Some bacterial protein probably facilitates the DNA pairing during recombination. It causes the local unwinding of dsDNA of the recipient cell from the 5′ end. Base pairing that is synapsis occurs between the homologous donor ssDNA and the recipient DNA. Unwinding of the recipient DNA continues at the end of assimilated DNA and allows the fraction of invading DNA to increase base pairs. This process is called branch migration . 

14. D). Integration- The endonuclease cuts the unpaired free end of donor DNA or the recipient DNA. This process is called trimming. The nick is sealed by DNA ligase . Consequently, a heteroduplex region containing a mismatched base pairs is formed . If the mismatch repair occurs again, it depends whether the unpaired base in the donor or recipient strand is removed. After replication the heteroduplex forms the homo-duplexes, one of these is of normal type and the second is transformed duplex. The donor genes differing from the recipient genes by a single base pair create a mismatch when integrated initially. The hex mismatch repair system (with LE markers) can correct either of donor strands. These two types of cells can be differentiated through plating method by using the antibiotic markers.

15. Methods of Transformation Transformation can be carried out in two methods; 1. Physical Methods 2. Chemical methods Physical methods include – -Electroporation - Microinjection - Biolistic – Gene gun - Ultra sonication Chemical methods – - Calcium chloride - PEG ( Polyethylin glycol) - DEAE ( Diethylaminoethyl cellulase )

16. Electroporation Electroporation is a process of changing the permeability of cell membrane of cells by  an electrical pulse that creates temporary pores for the uptake of macromolecules in the medium. It is done with an electrical instrument called electroporator which creates electric current and sends it through the electrodes of about 50-240 volts. The aluminum electrodes conduct the electric pulse through the medium in the cuvette. The electrodes are fixed on the inner side of the cuvette. The cell and DNA solution are mixed together and pipetted into the cuvette. This method is very effective for transfer of DNA to wide range of Gram-positive bacteria, and sometimes very high efficiency of transformation is achieved. Principle: The phospholipid bilayer of the plasma membrane has hydrophobic interior , any polar molecules , including DNA , are unable to pass freely through the membrane. The concept of electroporation capitalizes on the relatively weak nature of the phospholipid bilayer’s hydrophobic interior & hydrophilic exterior and its ability to reassemble after disturbance spontaneously.

17. Calcium Chloride (CaCl2) Calcium chloride transformation technique is the most efficient technique among the competent cell preparation protocols. It increases the bacterial cell’s ability to incorporate plasmid DNA, facilitating genetic transformation. When heat shock is provided the plasmid DNA passes into the bacterial cell presumably by affecting the physical state of the lipids in the membrane. To introduce rDNA into E .coli cells, the rDNA is added to the bacterial culture and the culture is treated with 50 mm calcium chloride (cacl2 ) solution at room temperature. Cacl2 adheres the rDNA onto the surface of E.coli cells and It modifies the bacterial cell wall to intake rDNA s. The bacterial culture is then heated gently up to 42ᴼc to induce E.coli cells to intake the rDNAs. In this method calcium chloride is used and can be performed in less than 3 hours. Exponential phase cells are harvested & treated with cold calcium chloride, which renders the cell competent or suitable for taking up DNA .

18. Ca2 + interacts with the negatively charged phospholipid heads of the cell membrane, creating an electrostatically neutral situation . Lowering the temperature stabilizes the membrane, making the negatively charged phosphates easier to shield. Then a heat shock creates a temperature imbalance and thus a current, which helps get the DNA into the cell.

19. Liposomes Mediated Transformation Liposome are fluid filled spherical vesicles made of phospholipids molecule. Produced from glycolipids, cholesterols, non-toxic surfactants and membranous proteins that were discovered in the year 1960 by British hematologist Dr. Alec D. Bangham . They can be preloaded with DNA by two common methods- membrane-membrane fusion and endocytosis thus forming DNA- liposome complex or lipoplex. This complex fuses with the cell membrane of target cell and it releases its contents into the cell. 

20. A liposomes are formed when lipids are agitated with water. It contains many concentric layers of phospholipids. In the liposome, polar heads face outward and the non-polar tails face to the Centre.  the rDNA , water and phosphatidyl choline are mixed together in a test tube and the tube is shaken well. During this process, lipid bilayers develop around the rDNA present in water and form a liposome. The liposomes in the tube are added to a culture of bacterial cells. Liposomes fuse with cell membrane and discharge their contents into the cells. As the inner lipid layer has nucleoproteins , cellular enzymes do not attack it . The inner lipid layer fuses with nuclear membrane and discharges its contents into the nucleus. The frequency of liposome fusion can be increased by the addition of polyethylene glycol (PEG)

21. 2. Conjugation. Conjugation is the transfer of DNA directly from one cell to another through cell-cell contact and usually involves the plasmids. It was the first extensively studied method of gene transfer and was discovered in 1946 by Joshua Lederberg and Edward Tatum when they observed genetic recombination between two different E . coli  strains that resulted in a wild type  E. coli  During conjugation, one bacterium serves as the donor of the genetic material, and the other serves as the recipient. The donor bacterium carries a DNA sequence called the fertility factor, or F-factor (F+). The F-factor is a circular double stranded DNA molecule of around 100 x 10 3  base pairs. The F-factor allows the donor to produce a thin, tube-like structure called a pilus, which the donor uses to contact the recipient. The pilus then draws the two bacteria together, at which time the donor bacterium transfers genetic material to the recipient bacterium. Typically, the genetic material is in the form of a plasmid, or a small, circular piece of DNA.  Conjugation helps to transfer plasmids which carry antibiotic resistance genes into the recipient bacteria.

22. Mechanism of Conjugation Conjugating bacteria are of two mating types:- Male types which donates their DNA, these are called F+ cells Female types which are recipient of DNA (F- cells) During Conjugation The F Pili of the F+ donor cell make contact with the F- recipient cell & pull the cell together. Rolling circle replication transfer one strand of the F factor into the recipient cell. Transfer of F factor is completed, yielding two F+ factor bacteria. Process of Conjugation In donor F+ cells, F factor may integrate into the host chromosome becoming Hfr (High Frequency of Recombination). Thus F+ cells become Hfr cells Conjugation between Hfr and F- cells results in replication of the chromosome with F factor. A single parental strand is transferred from Hfr cell to the F- cells. Only a small piece of F factor leads the chromosomal genes into F- cells Small strand containing chromosomal genes recombines with the DNA of F- cells. Thus F- cells receive only a part of chromosomal genes and hence do not get converted to F+ cells

23. 3. Transduction Transduction is the process by which bacteriophages transfers genetic material from one bacterium to another.  After multiplying, these viruses assemble and occasionally remove a portion of the host cell's bacterial DNA. Later, when one of these bacteriophages infects a new host cell, this piece of bacterial DNA may be incorporated into the genome of the new host. Bacteriophages were discovered by Norton Zinder and Joshua Lederberg in 1952. Bacteriophage Two types: 1 . Bacteriophage T4 2 . Bacteriophage λ 

24. Types of Transduction There are two types of transduction: 1. Generalized or Non- specialized transduction. 2. Specialized or restricted transduction. Generalized Transduction- A DNA fragment is transferred from one bacterium to another by a lytic bacteriophages while Carrying donor bacterial DNA due to an error . Specialized Transduction- A DNA fragment is transferred from one bacterium to another by a Temperate bacteriophages which carrying donor DNA along with Phage genome due to an error. In this case a phage inserts a genome at specific site.

25. Types of Bacteriophages. There are two types of bacteriophages; 1. lytic bacteriophages , 2. temperate or lysogenic phages . Lytic phage- This bacteriophage uses the lytic cycle for replication . In which the bacteriophage injects its genetic material into a host cell, allowing it to replicate producing many new phages. Once the host cell is filled with new bacteriophages, the host cell raptures from within, releasing the newly formed phages. Temperate or lysogenic phage The lysogenic cycle is one where a phage infuses its generic material into a host, but instead of rapidly replicating, this generic material finds its way to the host’s genetic material and infuses itself with it, becoming a prophage. It becomes part of the host’s genetic material and when the host cell divides, the temperate phage genetic material also undergoes a replication process.

26. Lytic & Lysogenic Cycles

27. Steps in Generalized Transduction 1 . A lytic bacteriophages adsorbs to a susceptible bacteria. 2 . The bacteriophages genome enters the bacterium where it directs the bacterium's metabolic machinery to manufacture bacteriophages components and enzymes. 3.Occasionally , a bacteriophages head assembles around a fragment of donor bacterium's nucleoid or around a plasmid instead of a phage genome by mistake. 4 . The bacteriophages are released. 5 . The bacteriophages carrying the donor bacterium's DNA adsorbs to a recipient bacterium. 6 . The bacteriophages inserts the donor bacterium's DNA it is carrying into the recipient bacterium. 7 . The donor bacterium's DNA is exchanged for some of the recipient's DNA.

28. Steps in Specialized Transduction Here Certain phages can transfer only a few restricted genes of the bacterial chromosomes (Only those bacterial genes adjacent to prophage in bacterial chromosomes Mediates the exchange of only limited numbers of specific genes Mediated by Bacteriophage λ It involves the following steps; 1.A temperate bacteriophages adsorbs to a susceptible bacterium and injects its genome. 2 . The bacteriophages inserts its genome into the bacterium's nucleoid to become a prophage. 3 . Occasionally during spontaneous induction, a small piece of the donor bacterium's DNA is picked up as part of the phage's genome in place of some of the phage DNA which remains in the bacterium's nucleoid. 4.As the bacteriophages replicates, the segment of bacterial DNA replicates as part of the phage's genome. Every phage now carries that segment of bacterial DNA. 5 . The bacteriophages adsorbs to a recipient bacterium and injects its genome. 6 . The bacteriophages genome carrying the donor bacterial DNA inserts into the recipient bacterium's nucleoid.

29. Applications of genetic transformation Through this transformation a non-virulent bacteria can be transformed into a virulent form It can be used in the laboratory for mapping of chromosomes of the bacteria. It can be used in bioremediation by introducing genes into bacteria giving them the ability to degrade hydrocarbons. In medicine it is used as gene therapy and drug delivery mechanism. It can be used in the production of human proteins such as insulin and clotting factors. It can be used in agriculture to improve crop disease resistance hence increase the yield. It is applicable in pharmaceutical industries for the development of vaccines.

30. summary Genetic transformation is the process through which microorganisms acquire foreign DNA material fragments from their surrounding and incorporate into their genome. The most commonly methods of gene transfer in prokaryotes are; Transformation Transduction Conjugation Transformation- This is the transfer DNA where the bacterial cell takes in DNA fragment from the environment which has been shed by a dead lysing bacteria into the surrounding. Transduction- This the transfer of DNA from one cell to another by the virus mostly the bacteriophage which infect the bacteria and take control of its replication machinery forming new virus particles. Conjugation- This is the transfer of DNA directly from one cell to another through cell-cell contact using conjugation tubes. The DNA transferred involves plasmids which can replicate in the bacterial cell independently of the chromosome.

31. conclusion Genetic transformation involves the up take of foreign naked DNA by the bacteria and incorporating it into its genome. Transformation can be natural or artificial and it occurs either through; transformation, conjugation or transduction. Genetic transformation improves diversity among the bacteria.

32. References Michael J Pelczar Jr. ECS Chan, Noel R Krieg, 2010Microbiology: An application Based Approach, Tata McGraw-Hill Education Pvt Limited New Delhi. Pg. 897. https://www.biologydiscussion.com/genetics/mechanism-of-transformation-with-diagram-genetics/65239