Gene Transfer in Bacteria
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Transcript of Gene Transfer in Bacteria
GENE TRANSFERTransformation, Transduction and Conjugation
Renz L. Salumbre, M.Sc.
Transfer of genetic material
Gene transfer is the movement of genetic information between
organisms
• Eukaryotes
• Essential part of the life cycle
• Sexual reproduction
• Gametes fuse to form zygote
• Each parent produces genetically different gametes
• Several genetic combinations transferred to offspring
Recombination is the combination of DNA from two
different cells
• In Bacteria, not an essential part of the life cycle
• Some genes of the donor cell are transferred to the recipient cell
• Resulting cell is called a recombinant
Rec proteins are essential to bacterial recombination
• Mutant genes: recA, recB, recC and recD
• Reduced recombinations
• RecA protein
• RecBCD protein - enzyme consisting of a polypeptide subunits encoded by other rec genes
Vertical Gene Transfer
• Parents to offspring
• Plants and animals
• In bacteria, asexual reproduction by binary fission
Bacteria pass genes to other microbes of the same generation
• Horizontal / Lateral Gene Transfer
• Transformation
• Transduction
• Conjugation
Significance of Gene Transfer
• Increase genetic diversity
• Mutations may account for some genetic diversity
• Environmental pressures lead to evolutionary changes
Transformation
• Frederick Griffith (1928)
• Pneumococcal infections in mice
• Natural transformation observed in Acinetobacter, Bacillus, Haemophilus, Neisseria, and Staphylococcus
• Also found in Saccharomyces cerevisiae
Mechanism of Transformation
• Naked DNA
• DNA released from an organism after the cell is lysed and the DNA no longer incorporated into chromosomes or other structures
• Organisms take up a maximum of about 10 fragments
Mechanism of Transformation• High cell density and depletion of nutrients
• Uptake of DNA
• Competence factor released into the medium
• Protein that facilitates entry of DNA
• Other factors
• Modifications of the cell wall
• Formation of specific receptor sites on the plasma membrane
• DNA transport proteins
• DNA exonuclease
Mechanism of Transformation
• DNA reaches the entry sites
• Endonucleases cut dsDNA into units of 7000-10000 nucleotides
• Strand separates and one strand enters the cell
• ssDNA vulnerable to nucleases
• Nucleases must be inactivated
• ssDNA base pairs immediately with a portion of the recipient chromosome
Mechanism of Transformation
• Donor ssDNA is positioned alongside the recipient DNA
• Identical loci are next to one another
• Enzymes in the recipient cell excise a portion of the recipient’s DNA and recombine it with the donor DNA
• Permanently part of the recipient’s chromosome
• Leftover DNA is broken down
• Number of nucleotides in the cell’s DNA remains constant
Mechanism of Transformation
Naturally transformable bacteria take up DNA from any source
With a few exceptions
Neisseria gonorrhoeae Haemophilus influenzae
Transduction
• DNA is carried by bacteriophage (phage)
• Discovered in Salmonella by Joshua Lederberg and Norton Zinder (1952)
Properties of Bacteriophages
• Composed of a core of nucleic acid covered by a protein coat
• Attaches to a receptor site on the cell wall of the bacterium
• Phage enzyme weakens cell wall allowing the passage of phage DNA
Type of pathway taken depends on type of bacteriophage
• Virulent phage causes destruction and death of a bacterial cell
• Phage genes direct the cell to synthesize phage-specific nucleic acids and proteins
• Destroy host DNA
• Other proteins and nucleic acids form phages eventually filling the cell up with it
• Phage enzymes rupture the cell
Type of pathway taken depends on type of bacteriophage
• Temperate phage does not cause a disruptive infection
• Phage DNA is incorporated into a bacterium’s DNA and is replicated with it
• Produces a repressor substance that prevents destruction of bacterial DNA
• Phage DNA does not direct synthesis of phage particles
• Replicate either as a prophage in a bacterial chromosome or by assembling into new phages
Lysogenic cycle
• Prophage - phage DNA incorporated into host bacterium’s DNA
• Lysogeny - persistence of a prophage without phage replication and destruction
• Known mechanisms to induce cells to enter lytic cycle
Bacteriophage Life Cycle
Transduction happens when some bacterial DNA is packed into the
heads of phages
• Generalized transduction
• Any bacterial gene can be transferred by the phage
• Specialized transduction
• Only specific genes are transferred
Lysogenic phages usually carry out specialized transduction
• Lambda (λ) phage in E. coli
• Inserts into specific locations during integration with a chromosome
• gal gene - galactose use
• bio gene - biotin synthesis
Specialized Transduction
• Cells containing lambda phage are induced to enter the lytic cycle
• Phage genes form a loop and are excised from the bacterial chromosome
• λ phage directs the synthesis and assembly of new phage particles and the cell lyses
• New phage particles released usually contain only phage genes; rarely does the phage contain one or more bacterial genes
Specialized Transduction by λ Phage in E. coli
Generalized Transduction
• Bacterial cell with phage DNA enter lytic cycle
• Phage enzymes break host cell DNA into many small segments
• Phage directs synthesis and assembly of new phage particles
• DNA packaged by the headful
• Bacterial DNA occasionally incorporated into phage particle; plasmids and DNA from other viruses may be incorporated
Generalized Transduction
Significance of Transduction
• Prophage DNA and host DNA demonstrate close evolutionary relationship
• Regions of similar base sequence
• Suggest viral origin of cancer
• Prophage can exist in a cell for long periods of time
• Malignant changes
• Animal viruses may have brought along genes from their previous hosts
• Provides a way to study gene linkage and chromosome mapping
Conjugation differs from transformation and transduction
• Requires contact between donor and recipient cells
• Transfers much larger quantities of DNA (occasionally, whole chromosomes)
• Discovered by Joshua Lederberg (1946)
Conjugation
• Plasmids are extrachromosomal DNA molecules
• Bacterial cells contain several different plasmids that carry genetic information for non-essential cell functions
• Conjugation involves
• Transfer of F plasmids
• High frequency recombinations (Hfr)
• Transfer of F’ plasmids
Characteristics of Plasmids
• Most are circular, double stranded extrachromosomal DNA
• Self-replicating
• F plasmid was first discovered
• Promiscuous cells
• Self-transmissible plasmids
• Conjugation with other species than their own kind
Functions of Plasmids
• F plasmids - synthesis of proteins that will assemble into conjugation pili
• Resistance (R) plasmids - genes that provide resistance to various antibiotics and to heavy metals
• Plasmids that direct the synthesis of bacteriocins
• Virulence plasmids that cause diseases
• Tumor-inducing (Ti) plasmids causing tumor formation in plants
Transfer of Fertility plasmids
• F+ and F- were found to exist in any population of E. coli capable of conjugating
• F+ cells contain Fertility plasmids
• F- lack F plasmids
• F plasmids carry information for the synthesis of F pilus (sex / conjugation pilus)
Transfer of Fertility plasmids
• DNA is transferred as a single strand via a conjugation bridge (mating channel)
• Sex pilus contains a hole that may permit the passage of ssDNA
• Evidences suggest that mating cells temporarily fuse during DNA transfer
Transfer of Fertility plasmids
• Pilus makes contact with a receptor site on surface of the F- cell forming a pore
• Inside the F- cell, pilus is pulled in and dismantled
• DNA from F+ cell enters F- cell
• Each cell synthesizes the complementary strand of DNA
• Both cells will become F+
High-frequency recombinations
• F+ strain that could induce 1000x more than the F+ x F- conjugations (L.L. Cavalli-Sforza)
• Hfr strains arise from F+ strains when F plasmid is incorporated into the bacterial chromosome
• Hfr cell is a donor in conjugation
• F plasmid initiates transfer of chromosomal DNA
• Only part of the F plasmid is transferred (initiating segment)
• Transfer of DNA occurs in a linear fashion with a precise time schedule (Wollman & Jacob)
• Recipient cell does not become F+
High-frequency recombinations
Transfer of F’ plasmids
• Process of incorporating an F plasmid into a bacterial chromosome is reversible
• DNA incorporated into a chromosome can separate from it and become an F plasmid
• Imprecise - can carry fragments of the chromosome
• F’ conjugate with F-
• Whole F’ plasmid is transferred
• Recipient cells have 2 of some chromosomal genes
Resistance Plasmids
• AKA R factors
• Formation of R plasmids are not due to antibiotics
• Use of antibiotics contribute to the survival of strains that contain R plasmids
• Organisms with R plasmids are said to be selected to survive
• Rapid process
• Large numbers of previously non-resistant organisms can become resistant quickly
R plasmids have two components
• Resistance Transfer Factor (RTF)
• DNA similar to F plasmids
• Implements transfer by conjugation of the whole R plasmid
• Essential for the transfer of resistance to another organism
• Resistant (R) genes
• One or more may be present
• Carries information that confers resistance
• Synthesis of an enzyme that inactivates the antibiotic
• Known resistance to sulfanilamide, chloramphenicol, tetracycline, streptomycin
Transposition
• R genes can move from one plasmid to another in a cell or even become inserted in the chromosome
• Transposable elements - mobile genetic sequence
• Insertion sequence contains gene that codes for an enzyme needed to transpose the insertion sequence
• Flanked by inverted repeats
• Replicate only when in plasmids or in a chromosome
Transposition
• Insertion sequence is copied by the transposase and cellular enzymes
• Copy randomly inserted into bacterial chromosome or another plasmid
• May cause mutations (spontaneous mutations)
• Transposons - transposable elements that contain genes for transposition
• Genes for toxin production or R genes