The following points highlight the four main modes of genetic recombination in bacteria during their reproduction in host cell. The modes are: 1. Transformation 2. Transduction 3. Conjugation 4. Sexduction.
Mode # 1. Transformation:
Transformation was first discovered by Griffith in 1928 in Diplococcus pneumoniae. In 1944, Avery and coworkers showed that DNA was responsible for transformation. In Transformation naked DNA molecules are taken in by bacterial cells. These DNA molecules replace homologous segments of the chromosomes of recipient cells; this may bring about a genetic change in these cells.
The uptake of DNA molecules is an active process requiring energy. Therefore, transformation occurs in only those species of bacteria that possess the enzymes for active transport. Further, only some cells in the cultures of these species are competent, i.e., capable of transformation. Competent cells contain a ‘competence factor’, which is most likely a cell surface protein involved in binding or uptake of DNA. In a given culture, the frequency of competent cells is the maximum during the late log phase.
The mechanism of transformation is summarised below; the process may be divided into the following five steps:
a. A reversible binding of the double-stranded DNA, donor DNA, to receptor sites on the surface of recipient cell.
b. An irreversible uptake of the donor DNA.
c. Degradation of one strand of the donor DNA molecules thereby making them single- stranded.
d. Integration (covalent insertion) of all or a part of the single-stranded donor DNA in the place of one strand of the homologous segment of chromosome of the recipient cell.
e. Segregation (due to semiconservative DNA replication) and phenotypic expression of the integrated donor gene or genes in the recombinant (transformed) cells.
Competent cells bind, take up and degrade one strand of DNA molecules from any species without any discrimination. But the integration of homologous DNA (DNA from the same species) is several orders of magnitude more frequent than that of heterologous (foreign) DNA.
Very small fragments of DNA are taken up, but a minimum length of about 500 base pairs is required for their integration. The degradation of one strand of the donor DNA is random in that either of the two strands is equally likely to be degraded. During integration, the donor DNA becomes physically inserted in the place of one strand of the corresponding segment of the chromosome of recipient cell.
Mode # 2. Transduction:
Transfer of DNA from one bacterial strain into another by a virus and the subsequent recombination between the chromosomes of recipient cells and the introduced DNA is known as transduction. The phenomenon was first described by N. Zinder and J. Lederberg in 1952. Transduction may be either (i) generalised or (ii) specialized.
a. Generalized Transduction:
In generalized transduction, a random or nearly random segment of the bacterial chromosome is transferred by the virus. This type of transduction is mediated by some virulent phages and by certain temperate phages whose chromosomes are not integrated at specific attachment sites in the host chromosome. Some of the mature particles of these phages contain a segment of the host chromosome.
These particles, called transducing particles, are responsible for transduction; they are produced during the lytic cycle of these phages. In some cases, transducing particles contain only bacterial DNA, while in others they contain both bacterial and phage DNA; the bacterial DNA segment may represent 1/100 to 1/50 of the total bacterial chromosome. Since all of the genes of the host chromosome are expected to be present in a population of transducing particles, this type of transduction is called generalized transduction.
Bacteriophages are called virulent or temperate on the basis of their interaction with their hosts. Virulent phages always multiply and lyse the host cells, which releases several progeny phage particles. But temperate phages may either (1) multiply or lyse the host cells like virulent phages (lytic cycle) or (2) their chromosome may become integrated into and behave as a part of the host chromosome (lysogenic pathway); an integrated phage chromosome is called prophage.
Chromosomes of temperate phages integrate into the host chromosome at one or a few specific attachment sites. A bacterium carrying a prophage is called lysogenic and the prophage-host relationship is known as lysogeny. The genes responsible for viral multiplication and lysis of host cells (lytic genes) are repressed in the prophages. A lysogenic bacterial cell is immune to further infections by the same virus as it turns off the lytic genes of the new phage chromosomes as well.
Only some virulent phages mediate transduction. Of these, E. coli phage P1, Salmonella phage P22, Bacillus subtilis phage PBS1 and SP10 have been used extensively for the mapping of mutant sites within individual genes or genes located in a short segment of the chromosome. The host DNA introduced by a transducing particle may become integrated in the double-stranded state in place of the homologous segment of the host chromosome, or it may remain free in the cytoplasm.
The former generates stable transduction, while the latter gives rise to abortive transduction. In abortive transduction, the nonintegrated DNA segment lying free in the cytoplasm is not degraded and is passed on to one of the two daughter cells at each cell division where genes express themselves. Cells carrying a nonintegrated DNA segment are partially diploid (merozygotes) and are used for complementation tests.
b. Specialized Transduction:
Temperate phage chromosome is integrated into their host chromosomes through a process of recombination. The recombination occurs at specific attachment sites present in both phage and host chromosomes. The attachment sites of the coliphage (phage of E. coli) chromosome, lambda (phage λ) chromosome (pp’), and E. coli chromosome (bb’) contain the following identical 15-base-pair sequence called the core sequence.
This site-specific recombination between E. coli and λ chromosomes occurs within this site, and is catalyzed by the product of int gene of λ in cooperation with another protein IHF (integration host factor). The λ chromosome is linear in mature phage particles. It changes into a covalently bound circular form after entering the host cell; this form may undergo integrative recombination with the host chromosome (Fig 9.4).
Prophages undergo spontaneous excision from the host chromosomes at very low frequencies (10–5/cell division). This process is generally very precise and site-specific. The free phage chromosome produced in this manner replicates and causes lysis of the host cell. The frequency of phage chromosome excision is greatly enhanced by certain treatments, e.g., irradiation with ultraviolet light; this is called induction.
During excision, the prophage chromosome is believed to loop out forming the figure of ‘8’ so that the two attachment sites lie next to each other. A site-specific recombination now occurs within the two attachment sites producing circular phage and host chromosomes (Fig. 9.4); in phage λ, this requires the product of phage genes int and xis plus protein IHF. This recombination is highly precise so that the excised phage chromosome is identical with the one that was originally integrated.
Occasionally, excision occurs at a site other than the original attachment site so that a portion of the phage chromosome is left in the host chromosome and a part of the latter is excised as a part of the phage chromosome. This gives rise to the specialized transducing particles. Such particles carry only those host genes that are located close to the site of prophage insertion. Therefore, specialized transduction transfers only those host genes that are closely linked with the phage attachment site.
For example, phage λ integrates between the genes gal (gene required for utilization of galactose) and bio (gene governing the biosynthesis of biotin) of E. coli (Fig. 9.4.), and it generally transduces only these genes. It should be noted that, of necessity, specialized transducing particles are produced at a low frequency (10–6 cells) by induction only; they are not produced by the natural lytic pathway of temperate phages.
When a specialized transducing particle infects a host cell, the phage chromosome (carrying a segment of the host chromosome) may integrate into the host chromosome through recombination at the attachment site. The host chromosome now contains the incomplete phage chromosome as well as the segment of host chromosome present in the transducing particle. Such a host cell, celled transductant or transduced cell is therefore diploid for the transduced segment (often called exogenote) as opposed to the host chromosome, which is known as endogenote.
Transductants having integrated phage chromosome, are known as primary transductions. They are usually unstable since they often lose the transduced DNA segment (exogenote) due to the excision of phage chromosome. The excised phage chromosome is unable to reproduce as it is incomplete, and many be diluted out through several cell divisions. Alternatively, the host DNA present in the phage chromosome may undergo recombination with the host chromosome transferring only some bacterial genes to the latter. This produces stable transductants since such cells rarely lose the transferred gene(s).
Mode # 3. Conjugation:
Conjugation in bacteria was discovered by J. Lederberg and E. Tatum in 1946; they were awarded the Nobel Prize in 1956. During conjugation, DNA is transferred from the donor cell (sometimes called male) to the recipient cell (also called female or F–) through a conjugation bridge formed between them. The male cells have specialized cell-surface appendages called F – pilli; their formation is governed by at least 13 genes present in a plasmid called F factor (‘fertility factor’, ‘sex-factor’, or ‘F plasmid’). The F factor is ~ 100 kb long and contains > 60 known genes.
It may either remain free giving rise to F+ male cells, or it may become integrated into the host chromosome producing Hfr (high frequency recombination) male cells. In free form, the F plasmid replicates using its own replication origin, oriV, and is maintained at a level of one copy per bacterial chromosome. But when F factor is integrated into the bacterial chromosome, oriV function is suppressed; the F factor DNA replicates as a part of the bacterial chromosome.
F factor can integrate into the bacterial chromosome through recombination at any one of the many sites. The integration of F factor is mediated by certain IS elements present in it and in the bacterial chromosome. When a male cell comes in contact with a female cell, it produces a conjugation bridge (formed possibly by protein TraD) through which DNA passes from the male into the female cell. When an F cell enters conjugation, only the F factor is transferred into the recipient cell converting the latter into an F+ cell.
Thus when some F+ cells are mixed with F– cells, all the cells in the culture becomes F+. In contrast, an Hfr male cell begins the DNA transfer with a part of the F factor followed by the bacterial chromosome. Generally, conjugation is spontaneously interrupted before the transfer of the entire Hfr chromosome so that the F– cell involved in conjugation rarely receives the complete F factor. As a consequence F– cells conjugating with Hfr cells ordinarily remain F– and are not converted into F+ cells.
The mechanism of transfer of both F factor (from F+ cells) and Hfr chromosome (from Hfr cells) appears to be similar. Protein TraY and/or Tral nicks one strand of the F factor or the Hfr chromosome at a specific site, called the ‘origin’ of transfer (oriT) in the F factor.
The 5′-end of nicked strand progressively separates from its complementary strand, and is transferred into the F– cell through the conjugation tube. This separation is brought about by TraY/Tral multimer, which binds to oriT after nicking. The separation is very rapid (~ 1200 bp per second) and does not depend on DNA replication.
Usually the nicked strand undergoes rolling circle replication. The single strand transferred into the F– cell serves as a template for the synthesis of its complementary strand, the DNA synthesis being discontinuous (Fig. 9.5). Further, the synthesis of complementary strand is simultaneous with the DNA transfer.
Since the cleavage occurs within the F factor, the sequence of DNA transfer from Hfr into the F– cells is as follows – a part of the F factor located away from the tra region, followed by the bacterial chromosome and finally the remaining part of F factor. Thus during Hfr by F– matings, F– cells acquire the complete F factor only rarely, and when the entire Hfr chromosome with its integrated F factor is transferred; in such cases, the F– cells become Hfr.
Conjugation is controlled by a ~33 kb region of the F plasmid called the transfer region (tr). This region contains ~40 genes, which are named as tra and trb loci (Fig. 9.6). These genes are arranged in 3 transcriptional units – (1) traJ encodes a regulator which turns on the other two units, (2) traM, and (3) a 32 kb transcription unit traY-I.
Gene finP is located on the opposite strand and encodes a regulator antisense RNA that turns off traJ; its activity requires the expression of another gene finO. The products of tra genes affect the properties of cell surface as well as are involved in DNA transfer; only 4 of the genes in the traY-I transcription unit are concerned directly with DNA transfer.
Gene traA encodes the monomeric protein pilin, which is assembled into F-pili; the assembly is governed by at least 12 tra genes. F-pili are 2-3 mm long ~8 nm diameter cylinders with a 2 nm axial hole. A typical F+ cell has 2-3 pili. Conjugation begins when the tip of F-pilus of a cell comes in contact with an F– cell.
Interaction of F-pili with F+/Hfr cells is prevented by two genes, traS and traT, which encode the so called ‘surface exclusion’ proteins that make the cell a poor recipient for such contacts. Incidentally, RNA phages and some single-stranded DNA phages attach to pili so that only male cells are sensitive to them. Once the mating is initiated, pili disassemble, the conjugating cells come closer and a conjugation bridge is formed either due to traD gene product alone or in combination with other gene products.
Protein TraM recognizes the formation of mating pair, following which TraY binds near oriT. This causes Tral to bind and nick oriT at a unique site called nic; it then binds to the 5′-end so generated. Tral is a relaxase; it also catalyzes unwinding of ~200 bp of DNA (helicase activity).
Usually, the 3′-OH at the nick is used for DNA replication simultaneous to unwinding, but this is not necessary for separation of the nicked strand. The separated single strand is transferred into the F– cell with the 5′-end first. It is important that only a unit length of F factor is transferred from an F+ to an F– cell even when it is linked with rolling circle replication (which can produce multimeric copies of the DNA molecule); the mechanism ensuring this is not known.
In E. coli, the transfer of complete Hfr chromosome takes ~100 minutes, depending on the strain, the rate of transfer being constant throughout a mating. The bacterial chromosome (exogenote) transferred into the F– cell is single-stranded; it undergoes replication to become double-stranded and then may undergo recombination with the chromosome of F– cell (endogenote).
It can be easily seen that two (or a multiple of two) simultaneous re-combinations between the exogenote and endogenote are required to transfer a segment of the former into the latter without affecting the structural integrity of the later [a single, or an odd number, of re-combination(s) would generate a linear or noncircular recombinant chromosome].
Thus the segment of Hfr chromosome transferred during mating can be determined by the recombinants recovered from the mating. The time required, after the initiation of mating, for the transfer of any two genes is used as an estimate of the distance between them. The E. coli linkage map is divided into 100 intervals, each interval equating one minute time required for gene transfer during mating. The 0 interval, the beginning of the linkage map, has been arbitrarily set at thrA gene.
The location of a gene in the chromosome is usually determined by interrupted mating experiments. In these experiments, the mating between Hfr and F– cells is interrupted at regular intervals after mating initiation and recombinants for the genes in question are scored in the progeny. Matings are easily interrupted by vigorously agitating the cell suspension in a blender as this breaks the conjugation bridges between Hfr and F– cells. The minimum time required for recombinants of the concerned genes to appear, gives the approximate location of the gene in the linkage map. Its exact location is then generally determined through transduction mapping.
The F factor can integrate at several sites in the bacterial chromosome; this determines the site of origin of transfer in the bacterial chromosome. The F factor may also integrate either in a clockwise (reading frame abcd) or in an anticlockwise (reading frame dcba) orientation; this determines the direction of bacterial chromosome transfer (i.e., clockwise or anticlockwise transfer). oriT is located at one end of the transfer region, and DNA transfer occurs away from transfer region into the bacterial chromosome. Therefore, following the transfer of a short leading sequence of F factor DNA, bacterial DNA is transferred.
Mode # 4. Sexduction:
The F factor is occasionally excised from Hfr chromosomes giving rise to F’ cells; the mechanism of excision is presumably similar to the excision of prophage X. Sometimes, error occurs during excision of the F factor so that a part of it is replaced by the bacterial chromosome (similar to the formation of specialized transducing particles).
Such F factor in which a part is replaced by the bacterial chromosome is called ‘F’ (F-prime) factors. F’ factors may carry from a single bacterial gene to about one-half of the bacterial chromosome. F’ factors are believed to be transferred to F– cells in the same manner as is the F factor.
Cells containing F’ factors are partial diploids (merozygotes) for the segment of host chromosome integrated in the F’ factor. These merozygotes are unstable as the F’ factor may be lost. Alternatively, recombination between an F’ factor and the bacterial chromosome may lead to the integration of the F’ factor (along with the bacterial genes contained in it) into the latter; such a gene transfer (mediated by F’ factor) is called sexduction or F-duction.
Thus sexduction produces partial diploidy for the segment of bacterial chromosome present in the F’ factor. Therefore, sexduction provides a unique opportunity for the determination of dominance relationship between alleles and of defining genes by complementation test. Since F’ factors are available carrying almost any segment of E. coli chromosome, sexduction analysis can be carried out with almost any mutation in the bacterial genome.
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