Genetic Recombination In Bacteria

Genetic recombination in bacteria became possible with the development of techniques that allowed the detection and investigation of bacterial mutations. These, in turn, enabled extensive research into the mechanisms of transfer of genetic information from one individual to another. Just as meiotic crossing over in eukaryotes, genetic recombination in bacteria forms the basis for methods of chromosome mapping.

This is a process whereby one or more genes on one bacterial chromosome are replaced with genes from another cell's chromosome of different genetic makeup. Although that is slightly different from the genetic recombination seen in eukaryotes, where there is a reciprocal crossing over, the end result is the same: genetic material has been transferred into and expressed by another cell and changed its genotype.

Three principal processes that allow genetic transfer among bacteria include: conjugation, transformation, and transduction. Overall, these permit inferences to be made about genetic diversity within a bacterial species and, in some cases, between different species. When the genetic transfer occurs between members of the same species, it is referred to as vertical gene transfer. On the other hand, when genetic transfer occurs between related but different species, the process is called horizontal gene transfer. Horizontal gene transfer has been an overwhelming driving power in bacterial evolution, and mostly genetic elements involved confer an advantageous survival ability to the recipient species. For example, genes resistant to antibiotics or one that enhances their pathogenicity is transferable from one species to another; in this regard, horizontal gene transfer has given cause for medical concerns. It has also been a key factor in bacterial speciation, since most bacterial species have acquired genes from other species.

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Conjugation in Bacteria

The Discovery of F+ and F- Strains

In 1946 Joshua Lederberg and Edward Tatum discovered bacterial recombination through a process called conjugation in which genetic material from one bacterium is transferred to another. They performed their initial experiments with two multiple auxotrophs (nutritional mutants) of E. coli strain K12. Strain A required methionine (met) and biotin (bio) to grow whereas strain B required threonine (thr), leucine (leu), and thiamine (thi). Neither strain was able to grow on minimal medium. After each strain had been grown in supplemented media separately, they mixed the cells and allowed them to grow together for several generations before plating them on minimal medium. Those few cells which were able to grow on this medium were selected as prototrophs.

The occurrence of spontaneous mutations in two or three independent locations at one time to revert these mutants to wild-type cells was highly unlikely; Lederberg and Tatum concluded that the prototrophs must have arisen through genetic exchange and recombination between the two strains. In this experiment, the rate of recovery of prototrophs was 1 in 10^7 cells plated. No prototrophs were recovered in the control experiments in which cells from strains A and B were plated separately. This led to the conclusion that genetic recombination had taken place.

Further research soon revealed that different bacterial strains are capable of transferring genetic material unidirectionally. When cells act as donors of chromosome parts, they are designated as F+ cells (F for fertility). The recipient bacteria, called F-, take up chromosome material from the donor and recombine it with their DNA. The "U-tube" experiment by Bernard Davis further supported the fact that cell-to-cell contact is necessary during the transfer of the chromosome, which proved physical contact is necessary for conjugation since no prototrophs were recovered in its absence. This is achieved through actual contact via a tubular projection called F pilus, through which adhesion and chromosome transfer between mating pairs take place.

Hfr Bacteria and Chromosome Mapping

In 1950, it was discovered that a special class of F+ bacteria, called Hfr (high-frequency recombination) cells, recombined 1,000 times more frequently than normal F+ strains. The Hfr strains showed a nonrandom pattern for the transmission of various genes within the bacterial chromosome. Although F+ strains transfer genes at random, those bacterial chromosomal genes were transferred in a specific order. However, that order was different for different Hfr strains. Scientists were able to use this nonrandom pattern of gene transfer as a means of mapping the E. coli chromosome.

In the mid-1950s, Ellie Wollman and François Jacob conducted experiments that defined the distinct difference between Hfr and F+ cells. Their work demonstrated that in conjugation, chromosome transfer in Hfr cells occurs with predictable timing and follows a pattern. Their findings helped establish chromosome mapping techniques in bacteria that gave further insight into the genetic structure and recombination processes in prokaryotes.

The interrupted mating technique showed that, with respect to the particular Hfr strain, some genes are transferred and recombined before other genes during bacterial conjugation. No recombination was detected after 8 minutes. At 10 minutes, the aziR gene was recombined, but other genes such as tonS, lac+, and gal+ were not transferred. By the 15-minute time, 50% of the recombinants were aziR and 15% were tonS, but none carried lac+ or gal+. At 20 minutes, lac+ began to appear, and at 25 minutes, gal+ was also transferred. This linear transfer of genes suggested that not only was the order of gene transfer predictable, but the distance between genes could be predicted from the time conjugation proceeded. It ultimately led to the construction of the first genetic map of the E. coli chromosome.

Wollman and Jacob extended their experiments employing different Hfr strains. They found that while the transfer of genes was always in a line, the order differed between strains. This difference was due to the variation in the origin, O, or first portion of the donor chromosome transferred during conjugation. The O site and the direction of gene transfer are determined by the integration point of the F factor. They further suggested that the E. coli chromosome is circular and the integration point of F factor varies in different strains, thus selecting different gene sequence transfer.

The genes near the O site were transferred first during conjugation; the last to be transferred was the F factor. However, conjugation usually did not continue long enough to transfer the entire chromosome, and that accounted for the fact that the recipient cells, when mated with Hfr cells, remained F-. One strand of the DNA of the donor entered the recipient, while the other strand replicated in the donor.

The interrupted mating technique also enabled the mapping of the entire chromosome of E. coli. Genetic recombination was observed to occur less frequently in F+ F- matings as compared to Hfr F- matings. This is because, in F+ * F-, gene transfer is random through a free circular form of the F factor. This may integrate the F factor into the bacterial chromosome. Such event turned an F+ cell to take up the Hfr state. This then resulted in random gene transfer events followed by low-frequency genetic recombination.

In 1959 Edward Adelberg discovered the F′ state in which the F factor carried several contiguous bacterial genes along with it. An F′ bacterium served like an F+ cell in transferring the F factor with chromosomal genes to an F-recipient, producing a partial diploid, now called a merozygote. Such merozygotes were extremely useful in studying bacterial genetic regulation.

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Bacterial Transformation

Transformation of bacteria refers to a gene process whereby liberated DNA, being released from the donor bacterium into the outside world, is engulfed and assimilated by the recipient bacterium. Such a process normally results in the manifestation of novel acquired genetic properties, allowing the recipient cell to acquire new characteristics.

Mechanism of Bacterial Transformation

Unlike conjugation or transduction, which are other processes of genetic transfer, bacterial transformation is not dependent on direct donor-recipient cell contact. Instead, it is merely based on the availability of free DNA in the environment. The recipient bacterium that successfully replicates and maintains the new genes is called a transformant.

Under conditions of environmental stress, some bacterial species actively excrete their DNA into the environment for uptake by competent cells. The competent cells control gene acquisition via a natural transformation process, which modifies their genetic composition according to environmental conditions.

Bacterial transformation is globally utilized in molecular biology and genetic engineering because it is efficient to introduce artificially manipulated DNA into cells of a receptor. The ability of bacterial transformation permits the DNA segment transfer to span from a single kilobase to dozens of kilobases, thereby allowing genetic investigation and biotechnology utilization.

Principle of Bacterial Transformation

DNA Uptake and Integration

The process of bacterial transformation relies upon the capacity of some bacteria to uptake and integrate exogenous DNA into their genetic material. Its efficiency is conditional on the recipient bacterium being competent, or capable of receiving naked DNA.

Natural and Induced Competence

Certain bacteria spontaneously become competent and shed DNA into the environment, especially towards the late stationary phase, by autolysis. But other bacteria like Escherichia coli do not become competent naturally but could be made to become so by artificial means.

Techniques for Increasing Bacterial Competence

1. Chemical Treatment (Calcium Chloride Method)

Enhances the permeability of the membrane to let the DNA enter the cell.

2. Electroporation

A high voltage electric field creates pores in the bacterial membrane for a short period, allowing the uptake of DNA.

3. Heat Shock Treatment

Sudden changes in temperature promote the entry of DNA into the bacterial cell.

Selection of Transformed Bacteria

Recipient bacteria transformed by the incorporation of new DNA can be selected after transformation by selectable markers. The markers can encode antibiotic resistance genes or metabolic functions that allow for bacterial growth under selective conditions.

Based on the type of transformation, the host DNA obtained is either integrated into the bacterial chromosome through homologous recombination or it remains as a plasmid that can replicate on its own without reference to the chromosome.

Steps of Bacterial Transformation

1. Development of Competence

There are certain bacteria that spontaneously develop competence under natural conditions, and others must be artificially induced through heat shock or electroporation.

2. DNA Binding to the Cell Surface

Free double-stranded DNA (dsDNA) adheres noncovalently to surface receptors of competent bacterial cells. This is a sequence-nonspecific process, and hence bacteria can absorb foreign DNA from unrelated organisms.

3. Processing and Uptake of DNA

Surface-associated dsDNA is cut by membrane-bound nucleases, leaving only a single-stranded DNA (ssDNA) fragment to enter the cell through a specialized DNA translocation channel.

4. Integration of DNA into the Chromosome

The incoming DNA can be subjected to homologous recombination, substituting a piece of the chromosomal DNA if there is enough sequence homology.

5. Plasmid DNA Maintenance

If the DNA is presented as a plasmid, it does not need to integrate into the chromosome and can be replicated autonomously.

6. Use of Selectable Markers

Selectable markers are used to detect transformed cells, typically by antibiotic resistance screening.

Types of Bacterial Transformation

Natural Transformation

Certain bacterial species have a natural competence for acquiring and incorporating environmental DNA.

Examples:

• Streptococcus pneumoniae

• Bacillus subtilis

• Neisseria gonorrhoeae

• Haemophilus influenzae

Artificial Transformation

Bacteria that lack natural competence may be made to accept DNA in the laboratory by methods such as:

• Chemical induction (CaCl₂ method)

• Electroporation

• Heat shock treatment

Example:

• Escherichia coli is one of the model organisms often used for artificial transformation because it is widely manipulated.

Examples of Bacterial Transformation

Frederick Griffith's Experiment (1928)

Transformation was first demonstrated in Streptococcus pneumoniae, where DNA from smooth (capsule-forming) strains was introduced into rough (non-capsule-forming) strains and converted them into virulent bacteria.

Neisseria and Haemophilus Species

These bacteria have species-specific DNA uptake through natural transformation.

Bacillus subtilis Transformation

A standard model organism for the study of the natural transformation process, B. subtilis actively takes up and inserts environmental DNA.

🗒 NOTE

Bacterial transformation is a basic operation in microbiology that makes genetic exchange and adaptation possible. Although certain bacteria have a natural ability to take up extraneous DNA, others are capable of being induced to transform through laboratory procedures. The process is of significant importance in genetic engineering since it enables researchers to introduce genes into bacterial cells for a myriad of purposes, such as medical research, industrial biotechnology, and drug development.

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Bacterial Transduction

Introduction to Transduction

Transduction is a mechanism of DNA uptake by bacteria in which donor DNA, consisting of fragments of bacterial chromosome, is introduced into the recipient’s cells via a bacterial virus (bacteriophage) vector. During this process, new genetic information may be acquired by the host cell.

Types of Transduction

There are two types of transduction:

• Generalized Transduction

• Specialized Transduction

In generalized transduction, the bacteriophages can pick up any portion of the host's genome. In contrast, with specialized transduction, the bacteriophages pick up only specific portions of the host's DNA.

Generalized Transduction

• During generalized transduction, virtually any bacterial gene can be transferred.

• The genetic transfer is mediated by virulent or lytic bacteriophage.

• Lytic bacteriophages are bacterial viruses that, upon infection of a host bacterial cell, destroy the DNA of the host and ultimately lyse the cell, releasing numerous viral progeny.

• Upon infection of a host cell by a lytic bacteriophage, viral enzymes degrade the host’s DNA into fragments.

• Viral DNA is not degraded because some of the bases in the bacteriophage genome are modified so that they are not recognized by viral enzymes.

• The viral DNA is then replicated, and viral proteins are synthesized.

• The newly replicated DNA is packaged into the coat proteins, and then infectious viral particles are assembled.

• When the viruses are fully assembled, viral enzymes degrade the cell’s envelopes, lysing the cell and releasing the viral progeny.

Defective Transducing Phages

• Infrequently, some of the host’s DNA is packaged into the virus along with an incomplete viral genome.

• When this happens, a generalized transducing phage is formed, which, although capable of initiating an infection, is unable to replicate itself or lyse the host cell.

• Some phage genes must be given up to accommodate the bacterial genes within the confines of the virus head.

• These defective transducing phages serve as vehicles for the transfer of host DNA (incorporated during viral assembly) from one cell to another.

• Because the packaging of host DNA into the viral particle is a random event, any given bacterial gene has an equal chance of being packaged and transferred to a recipient cell.

• Upon infection of a bacterial host cell by the transducing phage, the transducing DNA is introduced into the host’s cytoplasm and becomes incorporated into the bacterial genome by homologous recombination.

• The infected cell is not destroyed because the transducing phage is defective in that it does not have a full complement of genes.

Specialized Transduction

Specialized or restricted transduction is a process whereby a lysogenic bacteriophage serves to transfer a specific gene at a high frequency.

• When lysogenic bacteriophages infect host cells, their DNA is incorporated into the host’s genome by site-specific recombination.

• Specialized transduction is carried out only by temperate bacteriophage (which have the ability to adapt lysogenic lifecycles) that undergoes a lysogenic cycle in a donor cell.

• At first, the temperate bacteriophage enters into donor bacteria and then its genome gets integrated with the host cell’s DNA at a certain location.

• The viral genome remains dormant and passes from generation to generation into daughter cells during cell division.

• The bacteriophage that follows a lysogenic cycle is known as a temperate phage.

• When such a lysogenic cell is exposed to certain stimuli such as chemicals or UV light, it causes the induction of the virus genome from the host cell genome and begins the lytic cycle.

Transfer of DNA in Specialized Transduction

• Upon induction from donor DNA, this phage genome sometimes carries a part of bacterial DNA with it.

• The bacterial DNA lying on the sides of the integrated phage DNA is only carried during induction.

• When such a bacteriophage carrying a part of donor bacterial DNA infects a new bacterium, it can transfer that donor DNA fragment into the new recipient cell.

• In specialized transduction, only those restricted genes situated on the sides of the integrated viral genome have a chance to enter the recipient cell.

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