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


• 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


• 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


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)


• 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


• 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

Gene Transfer in Bacteria

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