Horizontal gene transfer with gene mapping

HGT Conjugation transformation and transduction with mapping

1. Horizontal Gene Transfer With Gene Mapping Presented by, Anish chhillar Anu Chaudhary R Ajay Rajaram Salman Khan

2. INTRODUCTION Gene transfer is defined as the movement of genetic information between the organisms. There are two types of gene transfer methods. They are: Vertical gene transfer - Transfer of genes from parents to offsprings. Horizontal gene transfer - Transfer of genes between two independent organisms. Horizontal gene transfer is common in prokaryotes. There are three types of horizontal gene transfer in prokaryotes. They are: Transformation Transduction Conjugation

3. TRANSFORMATION Transformation is the uptake of a naked DNA molecule or fragment from the surrounding environment by a cell and the incorporation of this molecule into the chromosome of the recipient. Transformation was discovered by Fred Griffith in 1928 Frederick Griffith suggested that bacterial strains are capable of transforming themselves by some factors, which he termed “transforming principle”. Sixteen years later, Oswald Avery, Colin MacLeod and Maclyn M cCarty demonstrated that transforming principle was the DNA, which provided evidence that DNA is the genetic material of bacterial cell.

4. Griffith experiment Griffith worked on bacterium Diplococcus pneumonia which is associated with certain types of pneumonia. This bacteria occurs in two forms. The first form has smooth (s) cells which secrete a covering capsule made-up of polysaccharide causing the colonies to be smooth and shiny.This type of strain is virulent. It produces pneumonia in mice. The second type has rough (R) cells which lack the polysaccharide capsule and colonies appear rough. This type is non virulent and it does not cause pneumonia in mice.

5. Heat killed S strain somehow transformed the R strain to virulent form. This process was called “Transformation”. The agent that was responsible for transformation was called “transforming principle”

6. Process of Transformation The ability of recipient bacterium to take up DNA molecules from the surrounding environment and become transformed is known as competence. Transformation requires both the uptake of DNA from the surrounding medium and its incorporation into the bacterial chromosome or a plasmid. As a DNA fragment enters the cell in the course of transformation, one of the strands is hydrolyzed or degraded at membrane surface by exonuclease, whereas the other strand associates with proteins as it moves across the membrane. Once inside the cell, this single strand may pair with a homologous region and become integrated into the bacterial chromosome.

7. Gram positive vs gram negative The Gram positive bacterial cell binds dsDNA fragment and the uptake of DNA into the cytoplasm is random and any portion of the donors may be entered. In case of Gram negative bacteria, DNA uptake is associated with the formation of small membranous structure called transformasomes which protrude outside the cell. The transforming DNA is taken into these vesicles where it is then internalized into the cell. DNA uptake in Gram negative bacteria is not random, DNA from only closely related species can enter into the cytoplasm. DNA uptake requires the presence of specific sequence called Uptake sequence, an 11 bp sequence repeated many times in gram neg bacteria genome. DNA must have this sequence to be bound by a competent cell.

8. Types of Transformation Natural Transformation: In this type of transformation, the bacteria takes up the naked DNA without the outside help. This bacterium are naturally competent, i.e. they can take up DNA from the environment without any special treatment. Natural Transformation is very rare. Artificial Transformation: In this type of transformation, the bacterium are made competent by certain physical or chemical treatments for the uptake of the naked DNA. Various methods are used for increasing the competence which includes the treatment with calcium chloride, electroporation, liposome etc.

9. Conjugation Conjugation is the process in which DNA is transferred from a bacteria donor cell to recipient cell by cell-to-cell contact. Joshua Lederberg & Edward Tatum discovered the conjugation process in bacteria . They experimented with two auxotrophic strains of E.coli K12 . Any bacterium harboring a self-transmissible plasmid is a potential donor(male strains) , because it can transfer DNA to other bacteria. Bacteria that lack the self-transmissible plasmid are potential recipient . The F-type plasmids use a transfer system known as the Tra system.

10. Bernard 's U-tube experiment The evidence for cell-to-cell contact was provided by Bernard Davis who built a U shaped tube. Filter allowed only the solution to pass but not bacteria. There was no growth without the contact. It proved conjugation requires physical contact.

11. Mechanism of bacterial conjugation Bacteria requires some special genes for conjugation that involves the tra gene. Tra locus includes the pilin gene and regulatory genes, which together form pili on the cell surface, polymeric proteins that can attach themselves to the surface of F-bacteria and initiate the conjugation After the conjugation process there will be the two F+ cells.

12. Hfr conjugation When F-plasmid is integrated with chromosomal DNA then such bacteria is known as high frequency recombination (Hfr) bacteria. In the cross (conjugation) between Hfr cell and F- cell, frequency of recombination is very high but frequency of transfer of whole F-factor is very low. Hfr cell acts as donor while F- cell acts as recipient. In this conjugation, chromosomal DNA is always almost transfer from donor to recipient cell together with portion of F- factor. So, frequency of recombination is high.

13. F’ conjugation Bacteria in which contains F-factor and a part of chromosomal DNA integrated in it is known as F-prime bacteria. F’ cells are formed from Hfr cell during induction of F- factor from chromosomal DNA in which F-factor carries a portion of chromosomal DNA along with it. In the cross (conjugation) between F-prime (F’) cell and F- cell, frequency of recombination is high as well as frequency of transfer of whole F-factor is also high.

14. TRANSDUCTION •Transduction is the transfer of bacterial genes by viruses. It is a frequent mode of horizontal gene transfer in nature and is mediated by viruses. •Viruses are unable to replicate autonomously. Instead, they infect and take control of a host cell, forcing the host to make many copies of the virus. •Viruses that infect bacteria are called bacteriophages. Some phages are replicated by their bacterial host immediately after entry. After the number of replicated phages reaches a certain number, they cause the host to lyse, so they can be released and infect new host cells. These phages are called virulent bacteriophages and the process is called the lytic cycle .

15. •Other bacteriophages do not immediately kill their host. Many of these viruses enter the host bacterium and, instead of replicating, insert their genomes into the bacterial chromosome. •Once inserted, the viral genome is called a prophage. The host bacterium is unharmed by this, and the phage genome is passively replicated as the host cell’s genome is replicated. •These bacteriophages are called temperate bacteriophages and the relationship between these viruses and their host is called lysogeny . Bacteria that have been lysogenized are called lysogens .

16. •About 70 to 90% of the transferred DNA is not integrated but often is able to survive temporarily and be expressed. Abortive transductants are bacteria that contain the nonintegrated, transduced DNA. • Specialized transduction , the transducing particle carries only specific portions of the bacterial genome. Specialized transduction is made possible by an error in the lysogenic life cycle of phages that insert their genomes into a specific site in the host chromosome. When a prophage is induced to leave the host chromosome, excision is sometimes carried out improperly. The resulting phage genome contains portions of the bacterial chromosome (about 5 to 10% of the bacterial DNA) next to the integration site. •The transducing particle will inject bacterial genes into another bacterium, even though the defective phage cannot reproduce without assistance. The bacterial genes may become stably incorporated under the proper circumstances. Example of specialized transduction is E. coli phage lambda.

17. What is gene Mapping? Genome mapping is the process of describing a genome in terms of the relative locations of genes and other DNA sequences. In bacteria gene mapping can be performed using Horizontal gene transfer mechanism.

18. Mapping bacterial genes with interrupted conjugation Conjugation can be used to map bacterial genes by mixing Hfr and F - cells that differ in genotype and interrupting conjugation at regular intervals. The amount of time required for individual genes to be transferred from the Hfr to the F - cells indicates the relative positions of the genes on the bacterial chromosome. François Jacob and Elie Wollman, who first developed this method of gene mapping.

19. Donor Hfr cells - sensitive to the antibiotic streptomycin (genotype str s ); resistant to sodium azide ( azi r ) and infection by bacteriophage T1 ( ton r ); prototrophic to threonine ( thr + ) l e ucine ( leu + ); and able to break down lactose ( lac + ) and galactose ( gal + ). F - recipient cells that were resistant to streptomycin ( str r ); sensitive to sodium azide ( azi s ) and to infection by bacteriophage T1 ( ton s ); auxotrophic for threonine ( thr - ) and leucine ( leu - ); and unable to breakdown lactose ( lac - ) and galactose ( gal - ) Donor Hfr cells: Hfr str s thr + leu + azi r ton r lac + gal + Recipient F cells: F str r thr - leu - azi s ton s lac - gal -

20. At regular intervals, a sample of cells was removed and agitated vigorously in a kitchen blender to halt all conjugation and DNA transfer. The cells were plated on a selective medium that contained streptomycin and lacked leucine and threonine. The original donor cells were streptomycin sensitive (str s ) and would not grow on this medium. The F - recipient cells were auxotrophic for leucine and threonine and also failed to grow on this medium. Only cells that underwent conjugation and received at least the leu + and thr + genes from the Hfr donors could grow on the selective medium. All str r leu + thr + cells were then tested for the presence of other genes that might have been transferred from the donor Hfr strain. All of the cells that grow on the selective medium are str r leu + thr + ; so we know that these genes were transferred. The percentage of str r leu + thr + exconjugates receiving specific alleles (azi r , ton r , lac + , and gal + ) from the Hfr strain are plotted against the duration of conjugation .

21. The first donor gene to appear in all of these exconjugates (at about 9 minutes) was azi r . Gene ton r appeared next (after about 10 minutes), followed by lac + (at about 18 minutes) and by gal + (after 25 minutes). These transfer times indicate the order and relative distances among the genes.

22. Mapping By Transformation Genes can be mapped in bacteria by taking advantage of transformation. The relative rate at which pairs of genes are cotransformed indicates the distance between them: the higher the rate of cotransformation, the closer the genes are on the bacterial chromosome.

23. Transduction: Using Phages to Map Bacterial Genes In transduction, bacterial genes become packaged into a viral coat, are transferred to another bacterium by the virus, and become incorporated into the bacterial chromosome by crossing over. Bacterial genes can be mapped with the use of generalized transduction

24. References Textbook-Genetics a conceptual approach-Benjamin A pierce. Textbook-Prescott, Harley, Klein’s Microbiology-7th Edition