Gene transfer &Reproduction in Bacteria

Dr. Sanjeev Kumar

Assistant Professor/Scientist Plant Pathology JNKVV-Jabalpur

[email protected]

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Rapid Development in Bacterial Genetics Reason • • • • •

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Highly precise method of study. Extremely rapid growth and small size. Millions of bacteria are easily handled and held in a drop of liquid Variety of genetic mechanisms that operate in bacteria greatly facilitated genetics analysis. Proved easier to related the genetic phenomena to the structure and function of the biologically important macromolecules

Mechanisms of Genetic Transfer in Bacteria

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1. 2. 3. 4.

Transformation Conjugation Transduction Lysogeny They differs in the mode in which DNA is acquired by the recipient bacterium

Genetic Recombination : • When the DNA enters the recipients cells by any of the three methods, it can synapse with the homologus region of the recipient genome and undergo recombination to give new genome types. • The reciprocal product of the recombination process is broken down by DNAase enzyme.

History

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1. Conjugation: • Discovered by Laderberg & Tatum (1946) in Escherichia coli

• •

(strain K12) Reported Genetic Recombination in E. coli. Awarded Nobel prize for explaining the genetic mechanism.

2.Transformation: • Discovered by F. Griffith in 1928 in Pneumococcus

pneumoniae (Diplococcus)

3.Transduction • Discovered by Laderberg & Zinder in 1951 started looking for recombination in Salmonella typhimurium 4. Sexduction • Discovered by Jacob & Adelberg, 1959 5. DNA is a genetic material • Discovered by Avery, MacLeod &Mc Carty, 1994

Transformation •

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Strain used by Griffith in experiment with P. pneumoniae

Pneumococcus strain

Morphology

Colony

Virulent

Capsulated

Smooth

Avirulent

Non –capsulated

Rough

1. Virulent strain caused death of mice on being injected while the avirulent strain was harmless. 2. Heat killed virulent strain failed to kill the mice.

3. When the living avirulent strain and the heat killed virulent strains were mixed and injected , the mice died. How ? 4. Was the avirulent strain transformed into a virulent strain in assocaition with the dead virulent bacteria ? YES 5. Grifith isolated bacteria from dead mice which had character of the virulent strain and proved virulent on being injected into other mice. 6. This charcter was transmitted to the progeny as a heritable character. 7. Called this phenomena as transformation and suggested that the heat killed bacteria provided the transforming principle. 8. Transforming principle was the polysaccharides of the capsule . 9. Nucleic acid as bearers of heriditory characters were not known then

Transformation

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Experiment by Avery, MacLeod &Mc Carty Proved that DNA is a genetic material

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1. Identified transforming principle in 1944- DNA nd proved that

2. 3. 4. 5.

DNA is a genetic material They extracted DNA, polysaccharides and proteins form a killed bacterial cells in pure form and added each separately to the avirulent cultures . It was only DNA that brought about the ransformation. Protease and RNA degrading enymes could not prevent the transforming action whereas DNAase inactivated the transforming activity. This confirmed DNA as the transforming pricniple

Experiment by Avery, MacLeod &Mc Carty Transformation by DNA

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Mechanism of Transformation

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1. Fragment of DNA, present in medium , get absorbed on recipient cells and then enters the bacterial cells. 2. Inside the cell , it replaces the homologus part of bacterial genome. 3. This result in the development of new characters which pass on to the progeny cells as stable and heritable character. 4. Recipient cell should be competent having binding sites for DNA at the surface to take up the external DNA. 5. It must not have DNAase that degrade DNA 6. Temperature and cations (Ca & Mg ions) enhance the efficiency of transformation

Experiment by Laderberg & Tatum Suggesting Recombination in E. coli

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1. One parental strain requires factor A & factor B for growth and was resistant to streptomycin and sensitive to phage. 2. Other parent required supply of growth factors C & D and was streptocyclins sensitive and phage resistant. 3. Individually both the strains unable to grow on minimal media. 4. When the two strains were MIXED and then spread on the same minimal medium, colonies appeared and grew in the absence of any of the growth factors A,B,C or D. 5. How did they come out ? 6. Growth of colony suggests the development of recombinant bacteria which must be A+, B+, C+, D+ genetically . 7. When tested for Streptomycin and phage resistance , these markers also appeared in new recombinations.

Experiment by Laderberg & Tatum Suggesting Recombination in E. coli

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Mechanism of Conjugation Conjugation between F (male) &Female

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1. Physical contact was involved in E coli K12 strain. 2. Strains of E. coli show sexual differences , one acting as donor of genes (male ) and other as recipient of genes (Female). 3. Donor strains produce tubular F pilli which establish contact with recipient cell and serves as conjugation tube for the passage of F factor ( F plasmid) and not a chromosomal gene. 4. F factor is small circular DNA molecule which is autonomous and lies free in the cytoplasm. 5. In a conjugation between F+ and F- the bacterial chromosome in not involved. 6. Only a duplicate of the F factor passes into the female cell. 7. Recipient is converted into male cell , so , the sex in E.coli can be said to be infectious. 8. Proved by method called interrupted mating experiment

Mechanism of Conjugation Conjugation between F (male) &Female

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Result: Conversion of female (F-) into male (F+) due to transfer of the fertility factor F

Conjugation between high fertility male (Hfr) & Female cells resulting in production of recombinant

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• F-plasmid can live in two states–either free in cytoplasm or integrated with a bacterial chromosome. • When inserted in the ‘chromosome’ the F+ male becomes a Hfr male, ‘high fertility male’ . • Hfr is a new type of male which shows enormous increase in its frequency of recombination. ‘Super male of James Bond. _, • When a Hfr male conjugates with a female F , the genetic material is transferred . • However, female is not converted into a male as the F+ factor is not passed on . • Genetic material may replace homologues portions of female genome and brings about ‘GENETIC RECOMBINATION.

Conjugation between high fertility male (Hfr) & Female cells resulting in production of recombinant female

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Transduction

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1. When a bacteriophage (phage) replicates in a bacterial cell (donor) , a few of the progeny virions incapsidate a piece of host DNA , instead of, or in addition to their own DNA. 2. These viruses when infecting a recipient host cell, introduce the DNA of the host also. Depending on the nature of DNA introduced , there are two types of transduction 1. Generalized transduction 2. Specialized transduction

Any bacterial gene that is encapsulated during the lytic cycle can be transferred to the recipient and the phage genome contains only donor DNA

Only specific genes ( that lie adjacent to the inserted phage genome in the lysogenic bacteria) are transferred

Generalized Transduction

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1. During lytic cycle of virulent phage, the DNA of the bacterial host is degraded into fragments. 2. When the virions are formed during maturation the bacterial genes rather viral genes get encapsulated. 3. When these phages containing only bacterial genes infect new cells, they inject the bacterial DNA, which may integrate into the bacterial chromosome. 4. Such phages when contain bacterial DNA instead of viral DNA are called defective phages as they do not cause death of cells. 5. Generalized transduction is also brought about by temperate phages during their lytic cycle. 6. They contain DNA fragment of lysed bacterium on their own DNA and transmit it to recipient lysogenic cell. 7. The bacterial DNA brings about recombination by integration with the bacterial chromosme.

Specialized transduction

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1. Caused by temperate phages which integrate in the host chromosome. 2. Bacterial genes close to prophage can be transduced (transferred). 3. Lambada phage of E.coli has been studied extensively for the specialized transdution. 4. During lysogenic cycle the phage genome is integrated with the bacterial chromosome . 5. When lysogenic cycle enters the lytic cycle, the phage genome is excised from the bacterial chromosome. 6. Occasionally , one or more bacterial genes that were adjacent to the viral genome go with the viral DNA. 7. After lytic cycle, the new progeny of temperate phage containing the bacterial genes enter the lysogenic cell and bring about transduction

Sexduction

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