FUNDAMENTALS OF MICROBIOLOGY

Lesson 24. GENETIC RECOMBINATION SYSTEMS

Module 6. Bacterial genetics

Lesson 24

GENETIC RECOMBINATION SYSTEMS

24.1 Transformation

Transformation is gene transfer resulting from the uptake by a recipient cell of naked DNA from a donor cell. Certain bacteria (e.g. Bacillus, Haemophilus, Neisseria, Pneumococcus) can take up DNA from the environment and the DNA that is taken up can be incorporated into the recipient's chromosome. Bacterial transformation was discovered by Frederick Griffith in 1928. Griffith worked with the pneumococci that cause bacterial pneumonia.

24.1.1 Griffith's experiment

The transformation process was first demonstrated in 1928 by Frederick Griffith. He experimented on Streptococcus pneumoniae, bacteria that cause pneumonia in mammals. When he examined colonies of the bacteria on petri plates, he could tell that there were two different strains. The colonies of one strain appeared smooth. Later analysis revealed that this strain has a polysaccharide capsule and is virulent, that it, it causes pneumonia. The colonies of the other strain appeared rough. This strain has no capsules and is avirulent. When Griffith injected living encapsulated cells into a mouse, the mouse died of pneumonia and the colonies of encapsulated cells were isolated from the blood of the mouse. When living non-encapsulated cells were injected into a mouse, the mouse remained healthy and the colonies of non-encapsulated cells were isolated from the blood of the mouse. Griffith then heat killed the encapsulated cells and injected them into a mouse. The mouse remained healthy and no colonies were isolated. The encapsulated cells lost the ability to cause the disease. However, a combination of heat-killed encapsulated cells and living non-encapsulated cells did cause pneumonia and colonies of living encapsulated cells were isolated from the mouse. How can a combination of these two strains cause pneumonia when either strand alone does not cause the disease? The living non-encapsulated cells came into contact with DNA fragments of the dead capsulated cells. The genes that code for the capsule entered some of the living cells and a crossing over event occurred. The recombinant cell now has the ability to form a capsule and cause pneumonia. All of the recombinant's offspring have the same ability. That is why the mouse developed pneumonia and died.

Fig. 24.1 Griffith’s experiment

24.2 Factors Affecting Transformation

24.2.1 DNA size state

Double stranded DNA of at least 5 X 10⁵ daltons works best. Thus, transformation is sensitive to nucleases in the environment.

24.2.2 Competence of the recipient

Some bacteria are able to take up DNA naturally. However, these bacteria only take up DNA a particular time in their growth cycle when they produce a specific protein called a competence factor. At this stage the bacteria are said to be competent. Other bacteria are not able to take up DNA naturally. However, in these bacteria competence can be induced in vitro by treatment with chemicals (e.g. CaCl₂).

24.2.2.1 Natural competence

About 1% of bacterial species are capable of naturally taking up DNA under laboratory conditions; many more are able to take it up in their natural environments. Such bacteria carry sets of genes that provide the protein machinery to bring DNA across the cell membrane(s).
DNA material can be transferred between different strains of bacteria, in a process called horizontal gene transfer.

24.2.2.2 Artificial competence

Artificial competence is induced by laboratory procedures and involves making the cell passively permeable to DNA by exposing it to conditions that do not normally occur in nature.

Calcium chloride transformation is a method of promoting competence. Chilling cells in the presence of divalent cations such as Ca2⁺ (in CaCl₂) prepares the cell membrane to become permeable to plasmid DNA. The cells are incubated on ice with the DNA and then briefly heat shocked (e.g., 42°C for 30–120 s) thus allowing the DNA to enter the cells. This method works very well for circular plasmid DNA. An excellent preparation of competent cells will give ~108 colonies per microgram of plasmid. A poor preparation will be about 104/μg or less. Good, non-commercial preparations should give 105 to 106 transformants per microgram of plasmid. The method, however, usually does not work well for linear DNA, such as fragments of chromosomal DNA, probably because the cell's native exonuclease enzymes rapidly degrade linear DNA. Interestingly, cells that are naturally competent are usually transformed more efficiently with linear DNA than with plasmid DNA.

Electroporation is another method of promoting competence. In the method the cells are briefly shocked with an electric field of 10-20 kV/cm that creates holes in the cell membrane through which the plasmid DNA enters. This method is amenable to the uptake of large plasmid DNA. After the electric shock the holes are rapidly closed by the cell's membrane-repair mechanisms.

The efficiency with which a competent culture can take up exogenous DNA and express its genes is known as Transformation efficiency.

24.3 Steps in Transformation

During transformation, DNA fragments (usually about 20 genes long) from a dead degraded bacterium bind to DNA binding proteins on the surface of a competent recipient bacterium. Nuclease enzymes then cut the bound DNA into fragments. One strand is destroyed and the other penetrates the recipient bacterium. This DNA fragment from the donor is then exchanged for a piece of the recipient's DNA by means of Rec A proteins (Fig. 24.2).

Fig. 24.2(a) A donor bacterium dies and is degraded



Fig. 24.2(b) A fragment of DNA from the dead donor bacterium binds to DNA binding proteins on the cell wall of a competent, living recipient bacterium

Fig. 24.2 (c) The Rec A protein promotes genetic exchange between a fragment of the donor's DNA and the recipient's DNA

Fig. 24.2 (d) Exchange is complete

Significance - Transformation occurs in nature and it can lead to increased virulence. In addition transformation is widely used in recombinant DNA technology.

24.4 Conjugation: Bacterial Conjugation

Conjugation: Bacterial conjugation is the transfer of genetic material between bacterial cells by direct cell-to-cell contact or by a bridge-like connection between two cells.

Distinguishing characteristics of conjugation:

• DNA transfer requires cell-cell contact.
• DNA transfer occurs via a conjugal pore.
• DNA transfer occurs in one direction - from donor to recipient not vice versa.
• DNA transfer does not require protein synthesis in donor.
• DNA transfer requires energy in donor cell - primarily AT

Do bacteria possess any processes similar to sexual reproduction and recombination? The question was answered in 1946 by the elegantly simple experimental work of Joshua Lederberg and Edward Tatum, who studied two strains of E. coli with different nutritional requirements. Strain A would grow on a minimal medium, only if the medium were supplemented with methionine and biotin; strain B would grow on a minimal medium only if it were supplemented with threonine, leucine, and thiamine. Thus, we can designate strain A as met⁻ bio⁻ thr⁺ leu⁺ thi⁺ and strain B as met⁺bio⁺ thr⁻ leu⁻ thi⁻. Here, strains A and B are mixed together, and some of the progeny are now wild type, having regained the ability to grow without added nutrients.

Fig. 24.3 Lederberg & tatum experiment

Lederberg and Tatum plated bacteria into dishes containing only unsupplemented minimal medium (Fig. 4.3). Some of the dishes were plated only with strain A bacteria, some only with strain B bacteria, and some with a mixture of strain A and strain B bacteria that had been incubated together for several hours in a liquid medium containing all the supplements. No colonies arose on plates containing either strain A or strain B alone, showing that back mutations cannot restore prototrophy, the ability to grow on unsupplemented minimal medium. However, the plates that received the mixture of the two strains produced growing colonies at a frequency of 1 in every 10,000,000 cells plated (in scientific notation, 1 × 10−7). This observation suggested that some form of recombination of genes had taken place between the genomes of the two strains to produce prototrophs.

But Lederberg and Tatum did not directly prove that physical contact of the cells was necessary for gene transfer. This evidence was provided by Bernard Davis (1950), who constructed a U tube consisting of two pieces of curved glass tubing fuse at the base to form a U shape with a fritted glass filter between the halves (Fig. 24.4). The pores of the filter were too small to allow bacteria to pass through but large enough to allow easy passage of the fluid medium and any dissolved substances. Strain A was put in one arm; strain B in the other. After the strains had been incubated for a while, Davis tested the content of each arm to see if cells had become able to grow on a minimal medium, and none were found. In other words, physical contact between the two strains was needed for wild-type cells to form. It looked as though some kind of gene transfer had taken place, and genetic recombinants were indeed produced.

Fig. 24.4 Davis experiment

24.4.1 Discovery of fertility factor

In 1952, William Hayes demonstrated that gene transfer observed by Lederberg and Tatum occurred in one direction. That is, there were definite donor (F+) and recipient (F-) strains, and gene transfer was nonreciprocal. One cell acts as donor, and the other cell acts as the recipient. This kind of unidirectional transfer of genes was originally compared to a sexual difference, with the donor being termed ‘male’ and the recipient ‘female’. However, this type of gene transfer is not true sexual reproduction. In bacterial gene transfer, one organism receives genetic information from a donor; the recipient is changed by that information. In sexual reproduction, two organisms donate equally (or nearly so) to the formation of a new organism, but only in exceptional cases is either of the donors changed.

By accident, Hayes discovered a variant of his original donor strain that would not produce recombinants on crossing with the recipient strain. Apparently, the donor-type strains had lost the ability to transfer genetic material and had changed into recipient-type strains. In his analysis of this ‘sterile’ donor variant, Hayes realized that the fertility (ability to donate) of E. coli could be lost and regained rather easily. Hayes suggested that donor ability is itself a hereditary state imposed by a fertility factor (F). Strains that carry F can donate, and are designated F⁺. Strains that lack F cannot donate and are recipients. These strains are designated F⁻.

24.4.2 F+ conjugation

Genetic recombination in which there is a transfer of an F⁺ plasmid (coding only for a sex pilus) but not chromosomal DNA from a male donor bacterium to a female recipient bacterium involves a sex (conjugation) pilus. Other plasmids present in the cytoplasm of the bacterium, such as those coding for antibiotic resistance may also be transferred during this process (Fig. 24.5).

Fig. 24.5 Diagrammatic presentation of F⁺ conjugation

24.4.3 Hfr conjugation

Genetic recombination in which fragments of chromosomal DNA from a male donor bacterium are transferred to a female recipient bacterium following insertion of an F⁺ plasmid into the nucleoid of the donor bacterium (Fig.24.6).

Fig. 24.6 Diagrammatic presentation of Hfr conjugation

24.4.4 Resistant plasmid conjugation

Genetic recombination in which there is a transfer of an R plasmid (a plasmid coding for multiple antibiotic resistance and often a sex pilus) from a male donor bacterium to a female recipient bacterium (Fig.24.7).

Fig. 24.7 Diagrammatic representation of resistant plasmid conjugation

24.5 Transduction

Transduction is the process by which DNA is transferred from one bacterium to another by a virus. It also refers to the process whereby foreign DNA is introduced into another cell via a viral vector. This is a common tool used by molecular biologists to stably introduce a foreign gene into a host cell's genome. When bacteriophages (viruses that infect bacteria) infect a bacterial cell, their normal mode of reproduction is to harness the replicational, transcriptional, and translation machinery of the host bacterial cell to make numerous virions, or complete viral particles, including the viral DNA or RNA and the protein coat (Fig.24.8).

Fig. 24. 8 Mechanism of transduction

24.5.1 Lytic and lysogenic (temperate) cycles

Transduction happens through either the lytic cycle or the lysogenic cycle. After a bacteriophage (or phage, in brief) enters a bacterium, it may encourage the bacterium to make copies of the phage. At the conclusion of the process, the host bacterium undergoes lysis and releases new phages. This cycle is called the lytic cycle. Under other circumstances, the virus may attach to the bacterial chromosome and integrate its DNA into the bacterial DNA. It may remain here for a period of time before detaching and continuing its replicative process. This cycle is known as the lysogenic cycle. Under these conditions, the virus does not destroy the host bacterium, but remains in a lysogenic condition with it. The virus is called a temperate phage, also known as a prophage. At a later time, the virus can detach, and the lytic cycle will ensue.

24.6 Types of Transduction

• Generalized transduction
• Specialized transduction

24.6.1 Generalized transduction

A DNA fragment is transferred from one bacterium to another by a lytic bacteriophage that is now carrying donor bacterial DNA due to an error in maturation during the lytic life cycle.

During generalized transduction, a phage assumes a lysogenic condition with a bacterium, and the phage DNA remains with the chromosomal DNA. When the phage replicates, however, random fragments of the bacterial DNA are packaged in error by new phages during their production. The result is numerous phages containing genes from the bacterium in addition to their own genes. When these phages enter a new host bacterium and incorporate their DNA to the bacterial chromosome, they also incorporate the DNA from the previous bacterium and the recipient bacterium is transduced (Fig.24. 9). It will express not only its genes, but also the genes acquired from the donor bacterium.

Fig. 24.9 Steps of generalized transduction

24.6.2 Specialized transduction

A DNA fragment is transferred from one bacterium to another by a temperate bacteriophage that is now carrying donor bacterial DNA due to an error in spontaneous induction during the lysogenic life cycle (Fig.24.10).

When the phage DNA breaks away from the bacterial DNA, however, it may take with it a small amount of the bacterial DNA (approx. 5%). When the phage DNA is used as a template for the synthesis of new phage DNA particles, the bacterial genes are also reproduced. When the phages enter new bacterial cells, they carry the bacterial genes along with them. In the recipient bacterium, the phage and donor genes integrate into the bacterial chromosome and transduce the recipient organism. Specialized transduction is an extremely rare event in comparison to generalized transduction because genes do not easily break free from the bacterial chromosome.

Fig. 24.10 Steps of specialized transduction