Recombinations: Transformation, Transduction and Conjugation.
Recombination is the transfer of genetic material from donor to the recipient cell or from one to another replicon. Recombinations provide the regular exchange of genetic information between bacterial cells.
Recombinant bacteria acquire the genetic properties of both parental cells: the basic number of the recipient’s genes and a small amount of genes from donor.
The Molecular Mechanisms of Recombination
Donor DNA interacts with the recipient DNA to become integrated into genome of the recipient cell. This requires genes and proteins with specific functions that govern recombination.
RесА, recВ, reсС, recD genes encode the synthesis of specific enzymes (recombinases RecA, RecBCD) that promote recombination processes.
RecА is the multifunctional protein that is activated after DNA binding. It acts as DNA-helicase (unwinds DNA double helix) and destroys several repressors, which block recombination. It catalyzes DNA cross-structure rearrangement.
RecA mutations lower recombination incidence for more than 1000 times.
RecBCD nuclease, encoded by recВ, reсС, recD genes, splits one DNA strand, allowing RecA binding. Also it accomplishes recombination by final cut of heteroduplex DNA cross-structure.
Recombination is based on the exchange of two complementary fragments between parental DNA molecules of donor and recipient cells. It includes incorporation or a donor DNA sequence into recipient one with the parallel transfer of the homologous recipient sequence backward into the donor DNA molecule. The fragment of DNA containing the complementary strands both from donor and recipient is termed as DNA heteroduplex.
At first, both of parental duplex DNA chains become unwound. Then they interact by their complementary DNA fragments, forming transition cross-like structure (so-called Holliday junction). The hydrogen bonds, maintaining DNA conformation, break in both parental molecules but lock again between primary and newly coming complementary DNA. The heteroduplex DNA fragment is formed, which carries the genetic sequences from donor and recipient DNA molecules.
Recombinases maintain the proper orientation and then split the complex of cross-reacting donor-recipient DNA strands.
Finally, DNA lygase links the free ends of phosphate backbone of recombinant DNAs thus restoring strands integrity.
Genetic recombinations in bacteria occur as the result of transformation, transduction, and conjugation.
Transformation is the direct uptake of donor’s DNA by the recipient cell.
F. Griffith discovered the process of transformation in 1928. He studied the experimental infection of mice triggered by the injection of the bacterial mixture, composed of a live decapsulated non-pathogenic type II Streptococcus pneumoniae and pathogenic capsulated type III S. pneumoniae, previously inactivated by heat. As the result, infected mice died due to septicemia caused by the virulent infection. F. Griffith found that type II S. pneumoniae acquired virulent properties being able to produce the capsule essential for S. pneumoniae type III. F. Griffith supposed that bacterial capsular polysaccharides were responsible for transformation.
In 1944 O. Avery, С. McLeod, and M. McCarthy revised the experiment of F. Griffith. They isolated transformational substance of high viscosity, resistant to proteases but sensitive to DNAse. It induced the transition of any type of pneumococci to type III S. pneumoniae. The substance was confirmed to be desoxyribonucleic acid. The scientists were the first who proved DNA transforming activity and demonstrated the role of DNA as a possible substance of heredity.
In nature the bacteria become able to capture the relatively large molecules of DNA only under special living conditions. These bacterial cells were designated as competent. Natural occurrence of this state is seldom among the bacteria. Some of them can undergo transformation only under the influence of competence factors, produced at a certain point of bacterial growth. This is followed by the marked changes of bacterial phenotype including the increased permeability of bacterial cell wall for nucleic acids and the expression of protein receptors on the membrane for DNA uptake.
The production of competence factors is not common among the bacteria; therefore, many bacterial species are poorly transformed.
Essentially competent bacteria can be found in different bacterial genera or species. Among them are Streptococcus pneumoniae, Neisseria gonorrhoeae, Hemophilus influenzae, Bacillus subtilis and others.
Many bacteria can be stimulated for transformation by external stimuli (temperature stress or calcium chloride exposure).
Electroporation is an artificial method to induce transformation of bacteria. Free DNA is added to bacterial cells and the electric current is applied. The electric current increases the permeability of the bacterial envelope (cytoplasmic membrane and cell wall) thus facilitating DNA uptake. Once appeared in the cytoplasm, DNA becomes incorporated into the recipient chromosome as the result of the homologous recombination.
Transduction is bacteriophage-stimulated genetic recombination in bacteria.
Transduction phenomenon was first described by N. Cinder and J. Lederberg in 1952. Bacteriophages as the specific viruses of bacteria were demonstrated to deliver genes from donor to the recipient bacterial cells. Phage genome may harbor genes encoding the resistance to antimicrobial agents, virulence factor synthesis (e.g., exotoxin and adhesin expression), flagella and pili formation, production of enzymes, etc.
The donor bacteria, the temperate phage, and recipient bacteria are the participants of the transduction process.
Three types of transduction have been revealed: general transduction, specific transduction and abortive.
As the result of general transduction the transfer of any bacterial gene may happen. The frequency of this rare genetic event is about of 10-4-10-7 per single phage particle. The incidence of general transduction can be arisen by pre-treatment of the phage with UV-light or other activators.
Specific transduction is performed by the temperate phage particles. They are generated after the excision of DNA sequence of the temperate phage from the nucleoid of bacterial lysogenic cells. It should be noted that lyzogenic bacteria have the genome with integrated DNA of temperate bacteriophage. When liberated from the nucleoid, phage DNA is further incorporated into capsids of nascent phage particles.
In case of specific transduction only definite gene clusters can be transduced (e.g., galactosidase locus, controlling the utilization of lactose in E. coli). After occasional non-proper excision, temperate bacteriophages can capture the bacterial genes flanking phage nucleic acid sequence. In that case the phage becomes defective but enables to transfer different host bacterial genes to other susceptible bacteria.
Abortive transduction occurs, when the genetic material delivered by the phage is not included into the genome of the recipient. It retains in the cytoplasm of the recipient cell. After the next cell division DNA of the phage remains non-replicated and stays only in one of the progeny cell, the second cell is free of phage DNA. Thus the phage genes become lost for the next bacterial generations. Abortive transduction is considered to be about 10 times more frequent event, than transduction types with integration of phage nucleic acid.
Transduction occurs between the bacteria of the same or different microbial species. Interspecies transduction has the evident biological value. Here bacteriophages enhance the diversity of living systems, thereby accelerating microbial evolution.
Conjugation is a one-sided transport of genetic material from one microbial cell to another by direct cell-to-cell contact.
Plasmid of a certain type (or, more correctly, episome) termed as F factor, or fertility factor, ensures the conjugation.
F factor replicates independently of nucleoid within bacterial cytoplasm.
Harboring F factor bacteria are the genetic donors, designated as F+ cells, whereas F─ сells are the recipients. They don’t contain F factor.
F plasmid of donor cell contains the genetic information for the synthesis of sex pili – special extracellular protrusions that promote binding of donor cell to the recipient bacteria. F plasmid also carries some additional genetic elements that is required for the successful transfer of DNA.
The transfer of F factor into the recipient cell takes place only in case of direct contact of the bacteria. F factor can exist in two forms: autonomous in bacterial cytoplasm and integrated into the bacterial nucleoid. Therefore, besides F+ donor cells, containing free F factor in cytoplasm, bacterial donors with integrated F factor sequence are found. They were designated as Hfr (high frequency of recombination) cells. These cells are characterized by essential high frequency of recombination (10-1-10-4), whereas the frequency of recombination between the F─ and F+ strains is in the range between 10-4 and 10-6.
Thus, there are major two variants of the conjugation.
In the first case autonomous F factor initiates the formation of the conjugation tube and reduplicates itself by the rolling circle mechanism. One linear strand of newly synthesized donor’s DNA is transferred into the conjugation tube. The recipient cell completes the structure of F factor’s DNA by synthesis of the novel DNA strand on the transferred donor’s DNA template. The remaining strand of F factor within the donor cell retains its circular form after duplication. As F factor copy has been delivered, the recipient cell becomes converted into the donor F+ cell.
Another variant of conjugation proceeds within Hfr cells. DNA sequence of Hfr cell is incised nearby the integrated F factor. But after the formation of conjugation tube the transfer of single-stranded linear DNA begins from the side of bacterial DNA localization. Thus F factor can be transported into recipient cell only after complete transference of nucleoid DNA. The latter is almost unlikely, so the recipient cell cannot obtain the properties of genetic donor.
Nevertheless, the nucleoid DNA fragment of the Hfr cell can be included in the genome of the recipient cell (F-) by recombination. As the result, an incomplete zygote (or merozygote) is formed that is composed of the whole genome of the recipient and some part of donor’s genome.
After conjugation both cells remain viable.
Similar to other recombinations, conjugation may occur not only between the cells of the same species, but among the cells from various species, thus leading to the production of interspecies recombinants.