BACTERIAL RECOMBINATION
As stated in, bacteria do not reproduce sexually like eukaryotic organisms. Their requirements of sexuality are met through certain alternate pathways (parasexual pathways) of genetic recombination which are called conjugation, transformation, and transduction.
Mediate transfer of genetic material (DNA) from one bacterial cell (donor) to the other (recipient) making possible the subsequent recombination events. The most obvious difference between these three processes is the mode of transfer of genetic material (DNA) from donor to recipient cell. In conjugation, donor cell transfers its DNA to a recipient cell only when both the cells are in physical contact through a specialized sex-pilus or conjugation tube. In transformation, there is transfer to cell-free or naked DNA from donor cell to recipient cell. In transduction, the transfer of genetic material (DNA) from donor cell to recipient cell is mediated by a bacteriophage.
The recombination in bacteria usually takes place (except for rare occurrence of complete DNA transfer during conjugation) forming partial normal diploids also called heterozygotes (heterogenotes) or merozygotes (merogenotes). Thus the recipient cells are converted to heterozygotes or merozygotes containing a fragment of the donor DNA, the exogenote, and a complete recipient DNA, the endogenote.
1. CONJUGATION
Lederberg and Tatum (1946) discovered conjugation in E. coli and its detailed studies were made by Woolman and Jacob (1956). Conjugation, as mentioned earlier, is a process by which genetic material is transferred from one bacterial cell (donor cell or male cell) to another (recipient cell or female cell) through a specialized intercellular connection called sex-pilus or conjugation tube. The maleness and femaleness of bacterial cells are determined by the presence or absence of F-plasmid (also called F-factor or sexfactor). F-plasmid, an extrachromosomal genetic material, is always present in the cytoplasm of donor or male cells, and the latter develop specialized cell surface appendages called F-pili or sex-pili under the control of F-plasmid. Recipient or female cells always lack F-plasmids and, therefore, F-pili are not present on their surface. F-plasmid or F-factor can exist in two different states: (1) the autonomous state in which it lies free in the cytoplasm and replicate independent of the bacterial chromosome (DNA); a donor or male cell containing F-factor in autonomous state is called F+ cell, and (2) the integrated state in which it is integrated (inserted) into the bacterial chromosome (DNA) and replicate alongwith it; a donor or male cell containing F-factor in integrated state is called Hfr cell (for high frequency recombination) or high frequency male cell. However, the recipient or female cell lacks F-factor and this is called F- cell.
1. a. Conjugation between a F+ (donor) cell and a F-(recipient) cell
In conjugation between a F+ (donor) cell and a F-(recipient) cell, it is the autonomous F-factor (F-plasmid) which is transferred, never the bacterial DNA. When the two cells (F+ and F-) come close to each other, the F- pilus of the F+ (donor) cell attaches with the F-(recipient) cell and acts as a conjugation tube. Simultaneously, the double-stranded circular F-factor DNA is nicked at a specific point, and begins to replicate producing a single-stranded copy of the F- factor DNA, which migrates through the tube into the cytoplasm of the F- (recipient) cell. It becomes doublestranded, and circular, and lies free in the cytoplasm thus rendering the recipient cell to become F+ donor cell. In this way, mixing a population of F+(donor) cells with a population of F+ (recipient) cells results in the conversion of virtually all the cells in the population becoming F+ (donor) cells.
1. b. Conjugation between Hfr Donor Cells and Recipient (F-) Cell
The Hfr donor cells are considered to be fertile because, unlike F+(donor) cells, their chromosomal segments are transferred from donor to recipient cells and the F- factor remains in situ.
When the two cells (Hfr and F-) come in contact, a conjugation tube develops between them. The circular DNA of Hfr donor cell is nicked and replication is initiated. The integrate F- factor always lies at the rear end of the DNA molecule. The replication of DNA starts towards the end near the conjugation tube and the newly synthesized single strand starts migrating through the tube into the recipient (F-) cell. In nature, the mating of two cells exists for a short period and gets interrupted resulting in the migration of only a portion of the donor DNA into the recipient cell. Since the F- factor lies at the rear end of the molecule, it is rarely transferred to the recipient cell.
1 | F- (Recipients) Cell |
2 | Hfr Donar Cell |
3 | F-Factor |
4 | Bacterial DNA with Integrated F-Factor |
5 | Bacterial DNA |
6 | F-Factor Lies at Rear end |
7 | Replicating Donar DNA (Migrates to Recipient Cell) |
8 | Synapsis of Homologous Parts of DNA |
9 | Part of Donor DNA Integrated with Recipinant DNA |
10 | Recombinant Cell |
11 | Replaced Aced Recipient DNA Degraded |
12 | Some Part of Donor DNA Transferred to Recipient Cell |
13 | Hfr Donor Cell |
The genes of the newly entered DNA fragment may replace the homolgous genes of the DNA of the recipient cell, resulting in a recombinant genetic material. The newly formed recombinant genetic material now possesses those male characters that have been transferred through recombination with the migrated DNA fragment.
1. c. Conjugation between F (F-prime) Male and F-(Recipient) Cell (Sex-Duction)
Existence of Hfr donor cells is not absolute. The F-factor integrated into the bacterial DNA of Hfr donor cells may dissociate and become free in the cytoplasm. The dissociation may be occasionally anomalous during which the dissociated F-factor may bring with it some genes of the bacterial chromosome. Adelberg and Burns (1958) first identified such a modified F- factor and called it F (F-prime) factor; the donor cell possessing this factor is called F (F-prime) male.
When a F male conjugates with F- (recipient) cell, the F-factor is transferred from donor to the recipient cell, and such a recipient bacterial cell becomes heterozygous (merozygous) for that part of the bacterial chromosome, which the F-factor had obtained during its anomalous dissociation. Transfer of F-factor to recipient cell apparently occurs by the same mechanism as F-factor, transfers during in F+ and F- mating and chromosome transfer in Hfr and F- cell mating. Genetic recombination of this type, mediated by F-factor, is called sex-duction or F-duction.
2. TRANSFORMATION
This process of genetic recombination was first studied by Griffith (1928), an English bacteriologist. He took two strains of the bacterium Streptococcus pneumoniae (= Pneumococcus pneumoniae), then called Diplococcus pneumoniae. One of the two strains was virulent or pathogenic and capsulated normal; it formed smooth colonies. The other strain was non-pathogenic or avirulent and non capsulated; it formed rough colonies on the culture medium. He experimented on mice as summarised below:
How Avirulent (nonpathogenic) strains transform into virulent (pathogenic) strains?
Virulent (pathogenic) strains of Streptococcus pneumoniae are capsulated whereas avirulent (nonpathogenic) ones are noncapsulated because they lack genetic information or capsule formation. When heat-killed (dead cells) of virulent strain are mixed with live cells of avirulent strain, the DNA containing the genes for capsule formation leak out of the dead virulent-strain cells and are taken up by the live avirulent strain cells. Recombination occurs and the progeny of the transformed cells of avirulent strain become capable of capsule formation, and, therefore, transform into virulent (pathogenic) strain.
It is obvious from
In transformation a free (naked) DNA molecule is transferred from a donor to a recipient bacterial cell. The donor bacterium undergoes lysis to free the DNA molecule and the recipient bacterium must be competent to receive it. This competence of bacterial cell is not a permanent feature; it has been demonstrated in relatively few bacterial genera and depends upon the growth phase of bacteria and the environmental conditions. When donor DNA comes into contact with the competent bacterial cell, it first binds on the cell surface and then is taken up inside the cell. In some of the cases, it is observed that the double-strand (ds) DNA enters inside the bacterial cell as such and its one strand is degraded by endonuclease enzyme therein leaving single-strand (ss) DNA whereas in others such as some species of Bacillus and Streptococcus it appears that only single -strand (ss) DNA enters the recipient bacterial cell.
An endonuclease enzyme now degrades one of the strands of dsDNA of recipient bacterial chromosome in corresponding region and this gap is filled by the donor ssDNA with the help of ligase enzyme which joins it with the DNA of the recipient bacterial chromosome. If the allelic forms of the donor and recipient genes are not identical, the donor DNA forms a heteroduplex with the recipient bacterial DNA. When the bacterial cell containing heteroduplex undergoes binary fission, the heteroduplex replicates forming two homoduplexes. One of these is a normal duplex which is all recipients in origin and the daughter cell containing it is like the recipient bacterial cell. The other homoduplex is a transformed duplex (hybrid genome) different from that of either the donor or the recipient bacterial genome. The daughter cell containing transformed duplex is a transformed cell and contains some of the characteristics of the donor bacterial cell which are inherited progeny to progeny.
3. TRANSDUCTION
This process of genetic recombination was discovered by Zinder and Lederberg (1952) in Salmonella typhimurium during their experiments with the objective of discovering whether E. coli type of genetic exchange also existed in S. typhimurium. In contrast to transformation, wherein free (naked) DNA is transferred, fragments of DNA are transferred from one bacterial cell to the other with the help of a viral carrier (bacteriophage) during transduction i.e., the transduction is a phage-mediated process of genetic material transfer in bacteria. The bacteriophage acquires a portion of the bacterial DNA of the host cell in which it reproduces and then transfers this acquired DNA to another bacterial cell to which it infects. Such bacteriophage is called transducing phage. Transduction is of the following two types: generalized (non-specialised) and specialized (restricted).
Why not all virulent phages bring generalized transduction?
As we know, the bacteriophages are classified into two types, virulent and temperate, on the basis of their interactions with the bacterial cell. Virulent phages always multiply and lyse the host cell after infection. Contrary to it, temperate phages may either (I) enter the lytic cycle like virulent phage, or, alternatively, they may (ii) enter the lysogenic-cycle during which their DNA are integrated into the bacterial chromosome. These are some virulent phages or those temperate phages that enter into the lytic-cycle and usually mediate the generalized transduction.
Some virulent phages it is said, because not all virulent phages mediate generalized transduction. For example, T-even bacteriophages (T2, T4, T6 ...) degrade the bacterial DNA and thus it is not available for packaging in progeny bacteriophage particle; in other cases the bacterial DNA is not degraded but it is too large to be packaged as such in the progeny bacteriophage particle.
3.1. Generalized Transduction
Transduction, which results in transfer of any bacterial gene from one bacterial cell to the other, is referred to as generalized or nonspecialized transduction. It is mediated by some virulent phages and certain temperate phages; E. coli phage P1, Salmonella phage P22, and Bacillus subtilis phages PBS1 and SP10 are such phages.
What happens when the injected phage-DNA does not integrate with recipient bacterial DNA during generalized transduction?
After a transducing phage injects its DNA into the recipient bacterial cell, the phage-DNA may either (i) be degraded by nucleases, or (ii) remain free in the cytoplasm, or (iii) integrate into the DNA of recipient bacterium. When the phage-DNA integrate into the bacterial DNA, it completes the process of transduction and such transduction is called complete generalized transduction. When the phage-DNA remains free in the cytoplasm, it does not replicate and is transferred as such to only one progeny cell during each cell division.
In generalized transduction, some of the developing progeny phages, during their normal lytic-cycle may accidentally acquire pieces of bacterial DNA. Such phages, after the lysis of the host bacterial cell and their release, attach to and inject their DNA into a new recipient cell but fail to re-establish lytic-cycle therein. Once inside the recipient bacterial cell, the injected DNA may be degraded by nucleases, in which case genetic exchange does not occur. The injected DNA, however, may undergo integration resulting in homologous recombination; as a result, the transduced cell may possess new combination of genes. The transduced bacterial cell now undergoes usual binary fission and produces progeny cells containing new combination of genes.
3.2. Specialized Transduction
In contrast to generalized (non-restricted) transduction, which results in transfer of any gene from donor to recipient bacterial cell, specialized (restricted) transduction is that which leads to the transfer of only specific (restricted) genes from donor to recipient cell. Specialized transduction is mediated by those temperate bacteriophages (e.g., lambda (l) phage, mu (m) phage and f80 phage) that usually incorporate (integrate) their DNA into the bacterial chromosome. The phage-DNA is called prophage in its integrated state with the bacterial chromosome; the bacterium having a prophage is said to be lysogenic, and this phage-host-relationship is called lysogeny.
Lysogenic temperate phages spontaneously switch over from lysogenic to lytic state at a low rate (about one in 195 cell divisions) in nature, or they may be induced to do so by irradiation with ultraviolet light. During this transition, the prophage is usually excised precisely from the specific site of integration in its exactly original form. But occasionally, it may excise imprecisely so that it takes with it that specific portion of bacterial chromosome which lies close to the site of prophage insertion and leaves a portion of its own DNA remaining integrated within the bacterial chromosome. Such prophage is called specialized transducing principle and is packaged into a developing phage particle inside the host bacterial cell. Phage particle so developed is called specialized transducing phage and is released after the host bacterial cell undergoes lysis.
Only those specialized transducing phages are viable that contain an amount of greater than 73% and less than 110% of the phage-DNA. When a viable specialized-transducing-phage infects a new bacterial cell, its specialized-transducing principle that already contains specific portion of bacterial chromosome inserts into the recipient bacterial chromosome thus making the latter diploid for that specific bacterial gene (partial diploid or heterogenote or merogenote). Since the specialized transducing phage is 'defective' phage as it has lost some genes during the excision, it functions in recipient bacterial cell only when the latter is already infected by another phage (termed as helper phage) that contains the missing genes.
3.3. Abortive Generalized Transduction
The genes located at the phage DNA may be expressed and synthesis of functional gene productions (e.g., enzymes) may be carried out in such cells. Cells carrying free-DNA of phage in their cytoplasm are called abortive transductants and the process of transduction is called abortive generalized transduction. It is considered that abortive generalized transduction is 10 to 20 times more frequent than complete generalized transduction. Abortive transductants are partially diploid and can be used to carry out complementation tests that provided the operational definition of the gene and helps determining whether different mutants are in the same gene or in different genes.
Thereby complements the lost phage-functions of the specialized transducing-phage. The partial diploid contains two copies of the concerned genes, one from donor bacterium and other from recipient bacterium, and is unstable. As a result, bacterial cells containing gene of donor bacterium and those containing gene of recipient bacterium segregate at a frequency of about one in 1,000 cell divisions.
For example, lambda (λ) phage integrates between the gal genes (required for the utilization for galactose as an energy source) and the bio genes (essential for the synthesis of biotin amino acid) in the E. coli chromosome. It transduces, therefore, only gal or bio genes thus making the recipient bacterial chromosome diploid for either gal or bio genes. Similarly, phage φ80 integrates near the trp genes (required for the synthesis of tryptophan amino acid) and transduces them.
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