Genetic recombination in_bacteria-jb

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conjugation, transformation, transduction in bacteria

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Genetic recombination in_bacteria-jb

  1. 1. Semester 1 Unit 2- Bacteria Reading Paper 2 Sexual Reproduction /Genetic Recombination in Bacteria:Transfer of DNA or genetic material from one bacterial cell (donor) to another cell (recipient). The transferreddonor DNA may then be integrated into the recipients nucleoid by various mechanisms and as a result it getsgenetically modified.Three types: a. Transformation, b. Transduction, c. ConjungationTransformation: Genetic recombination in which a DNA fragment from a dead, degraded bacterial cell entersa recipient bacterial cell, in liquid medium. Later the donor DNA integrates with the recipient DNA. Discoveredby Griffith in 1928 in Diplococcus pneumoniae.Confirmed by Avery, McCarthy and Mcleod in 1944.Involves 4 steps:1. A donor bacterium dies and its DNA gets degraded into fragments.2. A fragment of DNA from the dead donor bacterium binds to DNA binding proteins on the cell wall of a competent living recipient bacterial cell, present in the same medium3. The donor DNA enters into the recipient cell4. The entered donor DNA then integrates into the nucleoid or chromosomal DNA of recipient cell. As a result the recipient gets genetically modified.Transduction: Genetic recombination in which a DNA fragment is transferred from one bacterium to anotherby a bacteriophage. Bacteriophage (phage)are obligate intracellular parasites thatmultiply inside bacteria or viruses that infectbacteria. Discovered by Zinder andLederberg in 1952. There are two types oftransduction:Generalized transduction: A DNAfragment is transferred from one bacterium toanother by a lytic bacteriophage that iscarrying a piece of donor bacterial DNA dueto an error in the lytic life cycle. In this type,DNA segment from any part of the bacterialchromosome can be transferred. It occurs infollowing steps:1. A lytic bacteriophage adsorbs to a susceptible bacterial cell (donor cell)2. The bacteriophage DNA enters the bacterium and starts replicating. Viral capsid proteins are also produced.3. Meanwhile, the bacterial DNA is also broken into fragments.4. Viral DNA is packed inside capsids to form new phage particles. Occasionally, a fragment of bacterial DNA is inserted inside the capsid, instead of a phage DNA by mistake. Thus two types of phages are produced – one having viral DNA and other having extra bacterial DNA.5. The bacteriophages are released6. The released bacteriophage carrying the
  2. 2. donor bacterial DNA attaches and infects a new bacterial cell (Recipient Cell)7. The bacteriophage inserts the donor bacteriums DNA into the chromosome of recipient bacterium . Thus the recipient cell is genetically modified.Specialized transduction:This is mediated bytemperate phages whichinduce lysogenic life cycle.A specific DNA fragment(not any) is transferredfrom one bacterium toanother by such temperatebacteriophage.Six steps in SpecialisedTransduction:1. A temperate bacteriophage adsorbs and infects a susceptible bacterium (donor) and injects its DNA into the donor cell.2. The bacteriophage DNA is integrated into the bacterial nucleoid to become a prophage. In this condition, the bacterial cell multiplies.3. After some time, the phage DNA separates from bacterial chromosome. During this, a piece of bacterial DNA may remain attached to the phage DNA.4. The phageDNA replicates and the segment of bacterial DNA also replicates along with it.5. New phage particles are formed by assembly of phage DNA inside capsids. But now, every phage carries that segment of bacterial DNA.6. After release, when such phage particles infect new bacterial cells (recipient), they transfer the attached bacterial DNA to chromosome of recipient cells.Conjugation: Transfer of a segment of DNA, which usuallyinvolves a copy of plasmid DNA, from one living bacterial cell toanother through a physical connection is called conjugation.Discovered by Lederberg and Tatum in 1946 in E. coli. It is of 2types:F+ conjugation: Genetic recombination in which there is atransfer of a plasmid (but not chromosomal DNA) from aF+(male) donor bacterium to a F- (female) recipient bacterium.Involves a sex (conjugation) pilus. After conjugation, the F-(female) recipient is modified into F+(male) cell.Important steps:1. The F+ cell (male cell having a Plasmid DNA or Sex Factor and a Sex Pilus) serves as a genetic donor. Another The F- cell (female cell without Plasmid DNA or Sex Factor and a Sex Pilus) serves as a genetic receiver. These two cells approach each other.
  3. 3. 2. The sex pilus of the F+ cell adheres to the F- cell, forming a bridge like connection between two cells called conjugation canal. 3. The plasmid of the F+ cell replicates and one strand of the plasmid enters the F- cell, through the conjugation canal (There is no transfer of donor chromosomal DNA). As a result, F- is converted into F+. 4. The two cells separate from each other by the retraction of the conjugation canal. Hfr (High Frequency Recombination) Conjugation: Conjugation or Genetic recombination in which fragments of chromosomal DNA is also transferred along with plasmid DNA, from a F+ (male) donor bacterium to a F- (female) recipient bacterium following. Involves a sex (conjugation) pilus. Steps in Hfr Conjugation: 1. Some times, the F+ plasmid gets integrated into the chromosomal DNA of the F+ cell. Such cells are called Hfr males. 2. Conjugation occurs between one such Hfr cell and another normal F- cell. During this process, along with an incomplete copy of the integrated plasmid, some part of the chromosomal DNA of the Hfr cell is also transferred to F- cell. Since a complete F+ plasmid is not transferred, the recipient bacterium usually remains F- only.Gram Staining: The Gram stain is the most important and universally used staining technique in the bacteriologylaboratory. The method is named after its inventor,the Danish scientist Hans Christian Gram. Gramstaining is used to differentiate bacterial speciesinto two large groups called Gram +(positive) and Gram- (negative), based on thephysical properties of their cell walls.Procedure:1. Transfer a loopful of the liquid bacterial culture to the surface of a clean glass slide and spread over a small area. Slightly heat the slide so that the bacterial cells are fixed to the slide (Heat Fixing).2. Flood the slide with crystal violet (Primary Purple Stain) solution for up to one minute. Wash off Purple briefly with tap water (not over 5 seconds). Drain.3. Flood slide with Grams Iodine solution ( mordant) for about one minute. Wash off with tap water. Drain. Remove excess water from slide and blot.4. Flood slide with 95% alcohol (Decolorising agent) for 10 seconds and wash off with tap water. (Smears that are excessively thick may require longer decolorization. This is the most Purple sensitive and variable step of the procedure, Colorless and requires experience to know just how much to decolorize). Wash the slide.5. Flood slide with safranin (Counter Stain) solution for 30 seconds. Wash off with tap water. Drain and blot dry with blotting paper. Purple Do not rub. Pink
  4. 4. 6. Examine the slides under oil immersion objective (100X) of a microscope. Gram + cells appear Purple in colour while Gram- cells appear Pink.Principle of Grams Staining:Gram-positive bacteria have a thick mesh-like cell wall made of peptidoglycan (50-90% of cell wall) while Gram-negative bacteria have a thinner peptidoglycan layer (10% of cell wall). Gram-negative bacteria also have anadditional outer membrane which contains lipids, and is separated from the cell wall by the periplasmic space.• Crystal violet (CV) dissociates in aqueous solutions into CV+ and chloride (Cl – ) ions. These ions penetrate through the cell wall and cell membrane of both Gram-positive and Gram-negative cells. The CV+ ion interacts with negatively charged components of bacterial cells and stains all the cells purple.• Iodine (I – or I3 – ) interacts with CV+ and forms large complexes of crystal violet and iodine (CV–I) within the inner and outer layers of the cell. Iodine is often referred to as a mordant, but is a trapping agent that prevents the removal of the CV-I complex and therefore color the cell.• When a decolorizer such as alcohol is added, it interacts with the lipids of the cell membrane. A gram-negative cell will lose its outer lipopolysaccharide membrane and the inner peptidoglycan layer is left exposed. The CV–I complexes are washed from the gram-negative cell along with the outer membrane. Therefore they lose their purple colour and become colourless.• In contrast, a gram-positive cell becomes dehydrated from an ethanol treatment. The large CV–I complexes become trapped within the gram-positive cell due to the multilayered nature of its peptidoglycan. As a result, they retain their purple colour.• When counterstained with safranin , the decolorized and colourless gram-negative bacteria take the stain and turn pink or red. The already purple Gram+ cells do not take this stain. ©Dr. M Jayakara Bhandary Associate Professor Department of Botany Government Arts and Science College, Karwar - 581301

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