Horizontal Gene Transfer and Genetic Engineering Conjugation, Transformation, and Transduction

1. Horizontal Gene Transfer and Genetic EngineeringConjugation, Transformation, and Transduction Genetics

2. Gene Transfer • Refers to the movement of genetic information between organisms • When genes are transferred between two bateria or a bacteria and a virus it involves a combination of the DNA from two different sources. • This is referred to as recombination • This type of transfer is referred to as horizontal( lateral) gene transfer

3. Horizontal Gene Transfer • Horizontal gene transfer is a driving force in the development of drug resistance in bacteria • This type of transfer is different from the transmission of genetic chracteristics from one generation to generation vertically

4. Gene transfer can occur between bacteria and plants • Agrobacterium tumefaciens lives in the soil • It is able to transfer a plasmid from its cells into roots or stems through a scratch or injury to the plant tissue • The plasmid integrates in host DNA and affects the host to cause the growth of tumors called CROWN GALLS

5. Gene transfer from Bacteria to plant

6. Gene transfer can occur between viruses and animals • SV 40 • Simian virus • Is a DNA virus • Transforms or alters DNA and causes cancer • Used to study cancer and HIV

7. BCTERIAL CONJUGATION

8. Bacterial Conjugation • transfer of DNA by direct cell to cell contact • Contact with the pili • discovered 1946 by Lederberg and Tatum

9. F+x F– Mating • F+ = donor( contains the plasmid with the gene for conjugation) • This is referred to as the F factor or Fertility Factor • F– = recipient • does not contain F factor • F factor replicated by rolling-circle mechanism and duplicate is transferred ACROSS the pilus from the + to the - • recipients usually become F+ after it receives a copy of the DNA • donor remains F+

10. Gene transfer and recombination • Genes are transferred in a linear manner • The F factor integrates into chromosomes at different points

11. Mating • When two strains were mixed • There were incubated. • At intervals of 5 minutes, samples were taken of the F- cells • The cells were centrifuged so that they would know which genes were transferred. • The distance between genes was measured by the time that it took for the genes to be transferred. • During the first five minutes, the strains were mixed there was no recombination

12. F+x F– mating • In its extrachromosomal state the factor has a molecular weight of approximately 62 kb

13. Conjugative Proteins • Key players are the proteins that initiate the physical transfer of ssDNA, the conjugative initiator proteins • They nick the DNA and open it to begin the transfer • Working in conjunction with the helicases they facilitate the transfer of ss RNA to the F- cell

14. - Formation of Hfr

15. Hfr - high frequency of recombination

16. DNA Transformation • Uptake of naked DNA molecule from the environment and incorporation into recipient in a heritable form • Competent cell • capable of taking up DNA • May be important route of genetic exchange in nature

18. Transformation • Uptake of DNA can only occur at a certain cell density • Cells need to be in the log phase of growth • A competence factor is required for the uptake of DNA from the environment

19. Streptococcus pneumoniae nuclease – nicks and degrades one strand DNA binding protein competence-specific protein

20. Bacteria and transformation • Not all bacteria can be transformed in nature • Streptococcus pneumonia, Haemophilus influenza, and Neisseria gonorrhea

21. Lab protocol

22. Genetic recombination and transformation in the laboratory • Plasmids are designed to contain genes of interest • Transformation done in laboratory with species that are not normally competent (E. coli) • Variety of techniques used to make cells temporarily competent • calcium chloride treatment • makes cells more permeable to DNA

23. Cloning vectors

24. pAmp

25. pGlo and transformation

26. Bacteriophages Microbial Genetics

27. Horizontal gene transfer • Clearly this plays a central role in the diversity of E. coli • The greatest contributors are the bacteriophages • Among the 18 prophage remnants on O157 – 12 resemble lambda phage • They all contain a variety of deletions and or insertions • Some of the phages are so similar that they contain a 20 kb segment tat is identical.

28. Recombinant phages • It is believed that the phages have undergone recombination and diversification • They have been a major force in developing resistance and pathogenicity in bacteria such as E. coli and Streptococcus pyogenes • Recombination could occur with in a single cell • It could occur as the result of recombination

29. Bacteriophages

30. Bacteriophages • Bacterial viruses • Obligate intracellular parasites • Inject themselves into a host bacterial cell • Take over the host machinery and utilize it for protein synthesis and replication

31. T- 4 Bacteriophage • Ds DNA virus • 168, 800 base pairs • Phage life cycles studied by Luria and Delbruck

32. Bacteriophage structure

33. Bacteriophage structure(con) • Most bacteriophages have tails • The size of the tail varies. • It is a tube through which the nucleic acid is injected as a result of attachment of the bacteriophage to the host bacterium • In the more complex phages the tail is surrounded by a contractile sheath for injection of the nucleic acids

34. Bacteriophage structure • Many bacteriophages have a base plate and tail fibers • Some have icosahedral capsids • M13 has a helical capsid

35. Capsid • The base plate requires 12 protein products • The head or capsid requires 10 genes • The capside requires scaffolding proteins for assembly • DNA packaging a mysterious process • Many phages lyse their host cells at the end of the intracellular phase

36. T even phagesLuria and Delbruck • Four distinct periods in the release of phages from host cells • Latent period- follows the addition of phage( no release of virions) • Eclipse period – virions were detectable before infection and are now hidden or eclipsed • Rise or burst period – Host cells rapidly burst and release viruses • The total number of phages released can be determined by the burst size – the number of viruses produced per infected cell

37. General Steps

38. Steps in the life cycle • Adsorption of the virus to the host • This is mediated by tail fibers or some analagous structure • When the tail fibers make contact, the base plate settles to the surface • This connection which is maintianed by electrostatic attraction and the ions Mg++ and Ca++

39. Attachment • There is host specificity in the attachment and adsorption of the bacteriophage • There are receptors for the attachment. They vary from bacteria to bacteria • The receptors are on the bacteria for other purposes: the bacteriophages evolved to utilize them for their invasion

40. T even phages - Injection • The phage sheath shortens from 24 rings to 12 rings • The sheath becomes shorter and wider • This causes the central tube to push through the bacterial cell wall

41. Gp5 • The baseplate contains the protein gp5 with lysozyme activity which made aid in the penetration of the host

42. Early Genes • E. coli RNA polymerase starts transcribing genes( phage genes) within minutes of entering the bacterial cell • The early m RNA direct the synthesis of proteins and enzymes that are needed for hostile tack over • Some early virus specific enzymes degrade host DNA to nucleotides so that virus DNA synthesis can commence

43. Late mRNA • Phage structural structural proteins • Proteins that help with phage assembly • Proteins involved in cell lysis and release

44. Release • When the bacteriophages are released from the bacteria they can lyse the bacterial cell and break it open • They can be released through the cell membrane

45. Irreversible attachment • The attachment of the tail fibers to the bacterium is a weak attachment • The attachment of the bacteriophage is also accompanied by a stronger interaction usually by the base plate

46. Sheath contraction • The irreversible binding results in the sheath contraction

47. Injection • When the irreversible attachment has been made and the sheath contracts, the nucleic acid passes through the tail and enters the cytoplasm

48. Phage Multiplication Cycle – Lytic phages • Lytic phages or virulent phages enter the bacterial cell, complete protein synthesis, nucleic acid replication, and then cause lysis of the bacterial cell when the assembly of the particles has been completed.

49. Eclipse Period • The bacteriophages may be seen inside or outside of the bacterial cells • The phages take over the cell’s machinery and phage specific mRNA’s are made • Early mRNA’s are generally needed for DNA replication • Later mRNA’s are required for the synthesis of phage proteins

50. Intracellular accumulation phase • The bacteriophage sub units accumulate in the cytoplasm of the bacterial cell and are assembled