Bacterial geneticsChapter 8

9.1 Mutations and Mutants 9.2 Genetic Recombination 9.3 Genetic Transformation 9.4 Transduction 9.5 Conjugation9.6 Plasmids9.7 Transposons and Insertion Sequences 9.8 Comparative Prokaryotic Genomics 9.9 Genetics in Eukaryotic Microorganisms

• Microorganisms provide relatively simple systems for studying genetic phenomena and are thus useful tools in attempts to decipher the mechanisms underlying the genetics of all organisms.

• Microorganisms are used for the isolation and duplication of specific genes from other organisms, a technique called molecular cloning. In molecular cloning, genes are manipulated and placed in a microorganism where they can be induced to increase in number.

Section 1. Mutation and recombination

Mutation is an inherited change in the base sequence of the nucleic acid comprising the genome of an organism. Mutation usually brings about only a very small amount of genetic change in a cell.

Genetic recombination is the process by which genetic elements contained in two separate genomes are brought together in one unit. This mechanism may enable the organism to carry out some new function and result in adaptation to changing environments. Genetic recombination usually involves much larger changes. Entire genes, sets of genes, or even whole chromosomes, are transferred between organisms.

Section 2. Techniques of bacterial genetics: in vivo

In vivo : manipulate the genetic material within the organism

Genetic Transformation

Transduction

Conjugation

Section 3. Techniques of bacterial genetics: in vitro

In vitro: operate genetic material in the test tube

Restriction Enzymes

Molecular Cloning

Amplifying DNA: PCR

Auxotroph an organism that has developed a nutritional requirement through mutationCloning vector genetic element into which genes can be recombined and replicatedConjugation transfer of genes from one prokaryotic cell to another by a mechanism involving cell-to-cell contact and a plasmidDiploid a eukaryotic cell or organism containing two sets of chromosomesElectroporation the use of an electric pulse to induce cells to take up free DNAGene disruption use of genetic techniques to inactivate a gene by inserting within it a DNA fragment containing an easily selectable marker. The inserted fragment is called a cassette, and the process of insertion, cassette mutagenesis

WORKING GLOSSARY

Genetic map the arrangement of genes on a chromosome Genome the total complement of genes of a cell or a virus Genotype the precise genetic makeup of an organism Hybridization formation of a duplex nucleic acid molecule with strands derived from different sources by complementary base pairingMolecular cloning isolation and incorporation of a fragment of DNA into a vector where it can be replicatedHaploid a cell or organism that has only one set of chromosomesMutagens agents that cause mutationMutant an organism whose genome carries a mutation Mutation an inheritable change in the base sequence of the genome of an organism

Nucleic acid probe a strand of nucleic acid that can be labeled and used to hybridize to a complementary molecule from mixture of other nucleic acids Phenotype the observable characteristics of an organismPlasmid an extrachromosomal genetic element that has no extracellular form Point mutation a mutation that involves one or only a very few base pairs Polymerase chain reaction (PCR) a method used to amplify a specific DNA sequence in vitro by repeated cycles of synthesis using specific primers and DNA polymerase Recombination the process by which parts or all of the DNA molecules from two separate sources are exchanged or brought together into a single unit.

Restriction enzyme an enzyme that recognizes and makes double-stranded breaks at specific DNA sequencesShotgun cloning making a gene library by closing random DNA fragments Site-directed mutagenesis a technique whereby a gene with a specific mutation can be constructed in vitroSynthetic DNA a DNA molecule made by a chemical process in a laboratory Transduction transfer of host genes from one cell to another by a virusTransformation transfer of bacterial genes involving free DNATransposon a type of transposable element that carries genes in addition to those involved in transposition

Genetic Recombination

Genetic recombination involves the physical exchange of genetic material between genetic elements.

Homologous recombination results in genetic exchange between homologous DNA sequences from two different sources. This type of recombination is extremely important to all organisms. However, it is also very complex. Even in the bacterium Escherichia coli there are at least 25 genes involved.

A simplified version of one molecular mechanism of recombination. Homologous DNA molecules pair and exchange DNA segments.

The mechanism involves breakage and reunion of paired segments. Two of the proteins involved, a single-stranded binding (SSB) protein and the RecA protein.

Note that there are two possible outcomes, depending on which strands are cut during the resolution process. In one outcome the recombinant molecules have patches, whereas in the other the two parental molecules appear to have been cut and then spliced together.

In order to detect physical exchange of DNA segments, the cells resulting from recombination must be phenotypically different from the parents.

Detection of Recombination

Strains that lack some selectable characteristic that the recombinants will possess. For instance, the recipient may not be able to grow on a particular medium, and genetic recombinants are selected that can.

Various kinds of selectable and nonselectable markers (such as drug resistance, nutritional requirements, and so on) may be used.

Complementation

This can be determined by a type of experiment called a complementation test.Complementation was first used in diploid eukaryotic organisms

In prokaryotes genetic recombination is observed because fragments of homologous DNA from a donor chromosome are transferred to a recipient cell by one of three processes:

(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

Genetic Transformation

(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

Three main processes of genetic recombination in prokaryotes fragments of homologous DNA from a donor chromosome are transferred to a recipient cell

Conjugation

Transformation

Transduction

CompetenceA cell that is able to take up a molecule of DNA and be transformed is said to be competent.

Transformation

(a) Binding of free DNA by a membrane-bound DNA binding protein.

(b) Passage of one of the two strands into the cell while nuclease activity degrades the other strand.

(c) The single strand in the cell is bound by specific proteins, and recombination with homologous regions of the bacterial chromosome mediated by RecA protein occurs.

The introduction of DNA into cells by mixing the DNA and the cell

Transformed cell

The mechanism of bacterial transformation

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.

Concept

Transduction

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

And

Specialized transduction

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.

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.

Generalized transduction

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

Specialized Transduction

Genetic recombination involves the physical exchange of genetic material between genetic elements. It results in genetic exchange between homologous DNA sequences from two different sources.

Homologous recombination is extremely important to all organisms. However, it is also very complex .

Genetic Recombination

Direct contact between two conjugating bacteria is first made via a pilus. The cells are then drawn together for the actual transfer of DNA. Note the F-specific bacteriophages on the pilus

Bacterial conjugation (mating) is a process of genetic transfer that involves cell-to-cell contact.

conjugation

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 During Conjugation

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.

DNA Transfer in Bacteria

transformation

transduction

conjugation

(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

Three main processes of genetic recombination in prokaryotes fragments of homologous DNA from a donor chromosome are transferred to a recipient cell

Conjugation

Transformation

Transduction

Transformation

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

CompetenceA 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

(a) Binding of free DNA by a membrane-bound DNA binding protein.

(b) Passage of one of the two strands into the cell while nuclease activity degrades the other strand.

(c) The single strand in the cell is bound by specific proteins, and recombination with homologous regions of the bacterial chromosome mediated by RecA protein occurs.

The introduction of DNA into cells by mixing the DNA and the cell

Transformed cell

The mechanism of bacterial transformation

Artificially Induced CompetenceHigh 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.

DNA Transfer by Electroporationfor 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 arepresent outside the cells during the electric pulse, they can then enter the cells through these pores. This process is called electroporation.

TransfectionBacteria can be transformed with DNA extracted from a bacterial virus rather than from another bacterium, a process known as transfection.

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

And

Specialized transduction

Transduction

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.

Concept

:

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.

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.

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

Generalized transduction

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.

Generalized transduction

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

Specialized Transduction

Direct contact between two conjugating bacteria is first made via a pilus. The cells are then drawn together for the actual transfer of DNA.

Bacterial conjugation (mating) is a process of genetic transfer that involves cell-to-cell contact.

Conjugation

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.

Result of selected conjugation

Donor Recipient Molecules transferred Product

F+ F- F plasmid F+ Cell

Hfr F- Initiating segment of F plasmid and variable quantity of chromosomal DNA

F- with variable quantity of chromosomal DNA

F+ F- F+ plasmid and some chromosomal genes it carries with it

F+ Cell with some duplicate gene pairs: one on chromosom, one on plasmid

F-

F+

Hrf

F’

Cells possessing an unintegrat

ed F plasmid are called F', and strains that can act as recipi

ents for F' (or Hfr, see later in this section) are called F-.

F- cells lack the F plasmid;

F-

F’ Hrf

F+F+ x F-

Mechanism of DNA Transfer During Conjugation

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.