In prokaryotes several mechanisms viz., transformation, conjugation and transduction have been described that mediate the formation of new recombinants.
Transformation refers to transfer of relatively small segment of naked DNA from a donor cell (male) to the recipient (female) cell. Conjugation is the process of gene transfer between cells of opposite mating types that are in physical contact with each other. Transduction involves the transfer of genetic information from a donor cell to a recipient cell through a bacteriophage.
In the above processes not whole but a portion of chromosomes is transferred from a donor to recipient cell. Consequently the female cell becomes partially diploid called merozygote. The ultimate fate of the donated DNA is that whether it becomes incorporated in the recipient genome by recombination process degraded by restriction enzymes of the host or maintained as stable extra-chromosomal fragment.
1. Transformation:
The bacterial transformation was first discovered by Griffith (1928) between the two strains of Streptococcus (Diplococcus) pneumoniae which causes pneumonia in humans and mice. The results of Griffith are shown in Fig. 8.1. The RII strain (non-pathogenic, rough colony forming, mutant strain) did not cause death of mice, whereas the SIII strain (virulent and of smooth surface, wild strain) caused death of mice.
The heat killed SIII strain also showed results like RII strain. However, when heat killed SIII strain mixed with RII strain was injected in to mice, they died. This induction in change of RII strain was called transformation. Griffith thought that the transformation would have been caused by a protein.
Avery, Macleod and Mc Carty (1944) conducted the experiment and demonstrated that heat killed SIII strain transformed RII strain into virulent form which caused death of mice.
The transforming ability was not altered by treatment with enzyme or by RNase, but was completely destroyed by DNase. These findings showed that DNA has the ability to carry hereditary information. Major steps of transformation are shown in Fig. 8.2.
Subsequently, transformation has been shown in a number of bacteria such as Haemophilus, Neisseria, Xanthomonas, Rhizobium, Bacillus, Staphylococcus and Salmonella. Generally transformation does not occur in E. coli but it does after the treatment with calcium chloride. Possibly it facilitates the entry of DNA into the recipient cells.
Transfection:
Transfection is the process that involves transformation of bacterial cells with purified bacteriophage DNA resulting in production of the complete virus particles. The organisms not considered naturally transformable (e.g. E. coli. Salmonella typhimurium) can be transformed in vitro. Transformation can occur by using CaCl2 or electroporation (electric shocks) that brings about alteration in outer membrane.
In 1964, Foldes and Trautner were the first to show the infection of bacterial protoplast with purified nucleic acids and to this phenomenon they gave the term transfection. Since then transfection has been demonstrated in a number of bacteria such as B. subtilis, E. coli, H. influenzae, S. typhimurium, Staphylococcus, Streptococcus, etc.
When the transfected cells forming colonies on agar plates are lysed, clear zones are visible. These zones are called plaques. Notani and Setlow (1974) have discussed the mechanism of bacterial transformation and transfection. In recent years much work is being done on transfection in vitro for genetic engineering purpose. However, it has been an important factor in the success of researches in recombinant DNA technology.
2. Conjugation:
For the first time Joshua Lederberg and Edward L.Tatum (1946) in their brilliant and remarkable experiment presented the evidence for bacterial conjugation i.e. a process of transfer of genetic material by cell-to-cell contact.
They procured the two different auxotrophs (the mutant prototrophs lacking ability to synthesize an essential nutrient and, therefore, obtaining it or precursor from its surroundings) of E. coli, mixed them and incubated the two strains for hours in nutrient medium and plated on minimal medium (devoid of biotin, phenylalanine and other amino acids).
They used the double or triple auxotrophs to rescue from the chance of reversion. One strain (58- 161) required biotin (Bio–), phenylalanin (Phe–) and cystine (Cys–) for their growth, hence designated as Bio‑ Phe– Cys– Thr+ Leu+ Thi+. The second strain (W677) required biotin (Bio+), phenylalanine (Phe+), cystine (cys+), threonine (Thr–), leucine (Leu–) and thiamine (Thi–) and designated as Bio+ Phe+ Cys+ Thr– Leu– Thi–.
After incubation the recombinant prototrophic colonies (i.e. microbe requiring the same nutrients as the majority of naturally occurring microbial species) grew on minimal medium.
Production of recombinant prototrophs (i.e. Bio+ Phe+ Cys+ Thr+ Lei+ Thi+) would have been possible as a result of recombination) between the two auxotrophs (Fig 8.6).The recombinant prototroph had capacity to synthesize all the six growth factors i.e. amino acids.
Lederberg and Tatum (1946) could not give the proof for physical contact of cells required for gene transfer. The evidence for cell-to-cell contact was provided by Bernard Davis (1950) who built a U shaped tube. Two separate pieces of curved glass tubes were prepared and fused at the base to form a U shape with a fritted glass filter between the halves (Fig.8.7).
The filter allows the movement of media but not bacteria from both the ends of U tube. Nutrient medium was inoculated with different auxotrophic strain of E. coli. When it was inoculated the medium was pumped back and brought forth from the filter to facilitate the exchange of medium present on either sides of the filter.
The bacterial strains from both the halves of U tube were plated after 4 hour, of incubation onto minimal medium. The bacterial colonies did not appear on medium because cell-to-cell contact could not be established and, therefore, gene transfer did not occur. Hence, no recombinant prototrophs were produced.
Retro-Transfer:
Undoubtedly, William Hayes (1952) erected the scientific frame work of bacterial genetics, plasmid biology and horizontal gene transfer (conjugation) mechanism. Later on much work was done.
Thomas D. Brock (1990) has delineated two phases of research in bacterial genetics, pre-Hayes and post-Hayes. In his discovery of unidirectional transfer of genetic material, Hayes deduced the inequality between the strains, and identified 58-161 as a donor and W677 as a recipient.
However, during the mid-1980s, the universality of the unidirectional model of conjugation was questioned. IncP1, IncM and IncN plasmids were reported to mediate ‘back transfer’ or back mobilization from the recipient into the donor. This observation led to the birth of a novel conjugation phenomenon which is known as retro-transfer.
Retro signifies return or back and, therefore, retro-transfer must mean return transfer or back transfer. Retro-transfer implies only a behavioural conjugation rather than its molecular or genetic mechanism. It has also been demonstrated that in retro-transfer no novel mechanism of DNA transfer is involved. It is totally dependent upon the conversion of a female recipient to a male donor.
Top (1992) have proposed two mechanistically distinct models for retro-transfer, the one- step and the two-step. In the one-step (i.e. bidirectional) model, retro-transfer is a single event during which DNA moves freely in two directions between a cell bearing Tra+ plasmid and a cell carrying a Tra+ Mob+ plasmid.
Two step (i.e. unidirectional) involves two transfer events, the first event being the transfer of Tra+ plasmid from the donor to recipient, and the second step the transfer of the Tra+ Mob+ plasmid back to the original donor.
From the work done in subsequent years, the bidirectional model of retro-transfer emerged with the following characteristics:
(i) Retro-transfer is a one step process of bidirectional DNA transfer which consists of a single conjugative event during which DNA flows freely between donor and recipient,
(ii) Retro-transfer is mechanistically distinct from canonical conjugation and mobilization,
(iii) Retro-transfer is not dependent upon the transfer of the Tra+ plasmid to the recipient,
(iv) The time required for a retro-transfer is indistinguishable from that required for canonical conjugation,
(v) Retro-transfer is unaffected by surface exclusion, and
(vi) The ability to retro-transfer is a property possessed by an exclusive set of plasmid in compatibility groups.
3. Transduction:
The transfer of genetic material from one cell to another by a bacteriophage is called transduction. The phenomenon of transduction was first discovered by Zinder and Lederberg (1952) while searching for sexual conjugation in Salmonella species.
The infection by a bacteriophage is accomplished in several stages such as adsorption, penetration, replication, assembly, lysis and release. In brief the virus particles first attaches to specific receptor site on bacterial cell wall surface.
The genetic material penetrates the bacterial cell, and replicates independently by using cell machinery of the host. Consequently, the virus DNA is replicated into multiple copies, and synthesizes phage proteins. Complete phage particles are assembled and finally cell is lysed resulting in release of virus particles.
Depending on mode of reproduction the bacteriophages are of two types, the virulent phage and the temperate phage. The phages that reproduce by using a lytic cycle are called virulent phages because they destroy the host cell such as T phages, phage lambda (λ), etc.
In contrast the temperate phages ordinarily do not lyse the bacterial cell. The viral genome behaves as episome like F factor and becomes integrated into the bacterial chromosome.
The latent form of phage genome that remains within the host without harm and integrates with chromosome is called prophage. Bacteria containing prophage are known as lysogenic baceria and the relationship between phage and its host is called lysogeny.
The lysogenic bacteria can produce phage particles under some conditions, and the phage is able to establish the phenomenon of lysogeny and behaves as temperate phage. Usually, transduction occurs most readily between the closely related species of same genus of a bacterium i.e. intragenic. This preference is due to requirement for specific cell surface receptor for recognition of the phage.
In addition, inter-generic transduction has been shown between the closely related enteric bacteria such as between E. coli and Salmonella or Shigella species. Several genetic traits for example fermentation potential, antigens and chemical resistance are transducible.