Chapter8 Microbial genetics. 8.4 Plasmids 8.5 Genetic Conjugation, Transformation, transduction 8.6 Transposons and Insertion Sequences. 8.4 Plasmids. Circular genetic elements that reproduce autonomously and have an extra-chromosomal existence:. 1-1000 KB in size
Chapter8 Microbial genetics
8.5 Genetic Conjugation,
8.6 Transposons and Insertion Sequences
Circular genetic elements that reproduce autonomously and have an extra-chromosomal existence:
The presence of multiple antibiotic resistance is due to the fact that a single R plasmid contains a variety of genes coding for different antibiotic inactiviation enzymes
8.5 Three main processes of genetic recombination in prokaryotes fragments of homologous DNA from a donor chromosome are transferred to a recipient cell
(1) Transformation, which involves donor DNA free in the environment
(2) Transduction, in which the donor DNA transfer is mediated by a virus
(3) Conjugation, in which the transfer involves cell-to-cell contact and a conjugative plasmid in the donor cell
DNA Transfer in Bacteria
F plasmid of E. coli has the
special property of being able
to mobilize the chromosome
so that it can be transferred
during cell-to-cell contact.
Integration of an F plasmid
into the chromosome with the
formation of an Hfr. IS elements
are the sites of insertion.
Result of selected conjugation
Details of the replication
and transfer process
order of genes
Conjugation involves a donor cell, which contains a particular type of conjugative plasmid, and a recipient cell, which does not.
The genes that control conjugation are contained in the tra region of the plasmid (see Section 9.8 in your text ). Many genes in the tra region have to do with the synthesis of a surface structure, the sex pilus . Only donor cells have these pili,
The pili make specific contact with a receptor on the recipient and then retract, pulling the two cells together. The contacts between the donor and recipient cells then become stabilized, probably from fusion of the outer membranes, and the DNA is then transferred from one cell to another.
Mechanism of DNA Transfer DuringConjugation
A mechanism of DNA synthesis in certain bacteriophages, called rolling circle replication, was presented here to explains DNA transfer during conjugation .
if the DNA of the donor is labeled, some labeled DNA is transferred to the recipient but only a single labeled strand is transferred. Therefore, at the end of the process, both donor and recipient possess completely formed plasmids.
reverse mutation for the selected characteristic must be low.
This problem can often be overcome by using double mutants.
1. Bacteria need to be in a certain stage of growth.
2. Secrete a small protein called the competence factor that stimulates the production of 8 to 10 new proteins reauired for transformation.
1. A competent cell binds a ds DNA fragment
2. The DNA is cleaved by endonucleases to 5-15kb.
3. One stand is hydrolyzed by an envelop-associated exonuclease, the other strand associate with small proteins and moves through the plasma membrane.
4. Integration of transforming DNA
A number of prokaryotes have been found to be naturally transformable, including certain species of both gram-negative and gram-positive Bacteria and some species of Archaea. However, even within transformable genera, only certain strains or species are transformable
A cell that is able to take up a molecule of DNA and be transformed is said to be competent. Competence in most naturally transformable bacteria is regulated, and special proteins play a role in the uptake and processing of DNA. These competence-specific proteins may include a membrane-associated DNA binding protein, a cell wall autolysin, and various nucleases.
Competent cells bind much more DNA than do noncompetent cells as much as 1000 times more
Artificially Induced Competence
High efficiency natural transformation is found only in a few bacteria; Azotobacter, Bacillus, Streptococcus,, for example, are easily transformed. Many prokaryotes are transformed only poorly or not at all under natural conditions. Determination of how to induce competence in such bacteria may involve considerable empirical study, with variation in culture medium, temperature, and other factors
when E. coli is treated with high concentrations of calcium ions and then stored in the cold, the transformation by plasmid DNA is relatively efficient.
The introduction of DNA into cells by mixing the DNA and the cell
DNA Transfer by Electroporation
for artificial induction of competence are being supplanted by a new method termed electroporation.
Small pores are produced in the membranes of cells exposed to pulsed electric fields. When DNA molecules are
present outside the cells during the electric pulse, they can then enter the cells through these pores. This process is called electroporation.
The mechanism of bacterial transformation
Overview of events of
crown gall disease
following infection of
Mechanism of transfer
of T-DNA to the plant
1. Attachment (adsorption)
2. Penetration (injection)
3. Early steps in replication
5. Synthesis of protein subunits
6. Assembly and packaging
Viruses: Lysogeny and
Transduction involves transfer of host genes from one bacterium to another by viruses. In generalized transduction, defective virus particles randomly incorporate fragments of the cell's chromosomal DNA; virtually any gene of the donor can be transferred, but the efficiency is low. In specialized transduction, the DNA of a temperate virus excises incorrectly and brings adjacent host genes along with it; only genes close to the integration point of the virus are transferred, but the efficiency may be high.
In transduction, DNA is transferred from cell to cell through the agency of viruses. Genetic transfer of host genes by viruses can occur in two ways.
Generalized transduction: host DNA derived from virtually any portion of the host genome becomes a part of the DNA of the mature virus particle in place of the virus genome.
Specialized transduction: occurs only in some temperate viruses; DNA from a specific region of the host chromosome is integrated directly into the virus genome - usually replacing some of the virus genes.
Transduction has been found to occur in a variety of prokaryotes, including certain species of the Bacteria: Desulfovibrio, Escherichia, Pseudomonas, Rhodococcus,Rhodobacter, Salmonella, Staphylococcus, and Xanthobacter, as well as the archaean Methanobacterium thermoautotrophicum.
Not all phages can be transducer and not all bacteria are transducible
In generalized transduction, virtually any genetic marker can be transferred from donor to recipient
During a lytic infection, the enzymes responsible for packaging viral DNA into the bacteriophage sometimes accidentally package host DNA. This DNA cannot replicate, it can undergo genetic recombination with the DNA of the new host.
the DNA of lambda is inserted into the host DNA at the site adjacent to the galactose genes
On induction, Under rare conditions, the phage genome is excised incorrectly
A portion of host DNA is exchanged for phage DNA, called lambda dgal ( dgal means "defective galactose“ )
Phage synthesis is completed
Cell lyses and releases defective phage capable of transducing galactose genes
Bacteria can be transformed with DNA extracted from a bacterial virus rather than from another bacterium, a process known as transfection.
Insertion of a transposable
element generates a duplication
Both IS and Tn have short
inverted terminal repeats (IR)
at the ends of their DNA, IR are
involved in the transposition
How is the targeted
When a DNA segment is oriented in one direction, a particular gene is expressed. Whereas when it is oriented in the opposite direction, a different gene is expressed.