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In this article we will discuss about:- 1. Meaning of F-Plasmid 2. Organization of the F Plasmid 3. Mechanism of Transfer of F Factor 4. Integration of F Plasmid into the Bacterial Chromosome 5. F Mediated Transfer of Bacterial Chromosome 6. Conjugation Mapping 7. F-Duction (Sex-Ductiori) 8. Interspecific Transfer of Plasmids.
Meaning of F-Plasmid:
The F-plasmid is a conjugative plasmid found in several bacteria. The cell possessing the F factor is designated as “F+ cell” and acts as donor (male); a cell devoid of this factor is designated as “F– cell” and acts as recipient (female) during conjugation.
A part of the bacterial chromosome may also enter the recipient cell and lead to genetic recombination. Eventually the F factor gets transmitted from F+ cell into the recipient F– cell; such recipient (F–) cells therefore, become F+ cells.
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The F factor can exist independent of the host chromosome, or it may become integrated into it (Fig. 18.1). The bacterial cell in which the Factor is integrated into the chromosome is called an Hfr (high frequency recombination) cell. The F factor integration causes a relatively very frequent transfer of the bacterial chromosome from the donor into the recipient cell resulting in a high frequency of recombination.
In such a situation, the recipient ceil becomes diploid for a short period of time for the part of the donor chromosome transferred into it; such cells are therefore, called merozygotes, partial zygotes or merodiploids. Infrequently, the entire chromosome of the Hfr cell may get transferred into the F– cell; such F– cells convert into Hfr cells.
Organization of the F Plasmid:
The F plasmid is a double-stranded circular DNA molecule of about 100 kpb (105 nucleotide pairs). It is about 30 pm long which is about 1/40 of the length of the bacterial (E. coli) chromosome. The origin point of replication in F factor for transfer of DNA into the recipient cell is called “ori T” (Fig. 18.2), while the origin point at which replication of the F factor begins as an autonomous unit (during cell division of the F1” cells) is designated as “ori V” (Origin of vegetative replication).
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The transfer operon (tra) is a large region (33 kb) of the F plasmid. It is required for conjugation. The tra operon contains more than 25 genes which are required for transfer of DNA. Most of these genes are expressed coordinately as part of a 32 kb transcription unit. The genes tra J and tra M are expressed separately. The F factor tra operon has homology with the tra operon of R plasmid (F-like R plasmids). It also contains IS elements that provide the sites for its integration into the host chromosome.
Mechanism of Transfer of F Factor:
The F+ bacterial cells have surface appendages called F-pili or sex-pili which are coded by the F plasmid. An F+ cell has 23 pili. They are hair-like structures of about 2-3 µm length and appear as extension from the bacterial cell wall. A single subunit protein “pilin”, coded by gene tra A polymerizes into “pilus”.
The pilin is polymerized into a hollow cylinder of about 8 nm diameter and 2 nm axial hole. The sex pilus is necessary for contact between the donor and the recipient cell. These pili may also serve as specific attachment sites for certain icosahedral RNA bacteriophages (MS2, QB) and filamentous DNA bacteriophages (M13, f9).
When the tip of the F pilus contacts the surface of recipient (F–) cell, mating is initiated. Mating cannot occur between two F+ cells because the genes tra S and tra T code for “surface exclusion proteins”. The Tra Y and/or Tra I makes a nick in one strand of the F factor at the ori T point (origin transfer point). Then this protein complex (Tra Y/Tra I) binds to DNA and causes the unwinding of about 200 bp.
The protein complex moves along DNA from 5′-end and unwind DNA at the rate of 1200 bp per second. The 5′-end of the cut end enters the recipient cell and is released when the circle gets back to the origin, i.e., only the unit length of plasmid DNA is transferred.
If there is concomitant DNA replication the process becomes a rolling circle type. After the transfer is completed, a complementary strand is synthesized on the transferred DNA strand into the recipient cell, and the plasmid is circularized.
Integration of F Plasmid into the Bacterial Chromosome:
Integration of the F factor into the bacterial chromosome occurs at specific regions of DNA called insertion sequences (IS) (Fig. 18.2, 18.3). The F factor carries several IS regions, such as, IS 2, IS 3, V-δ. The E. coli chromosome carries 20 IS regions which are scattered throughout the chromosome with both, clock-wise and counter clock-wise orientations.
The IS elements provide the loci for homologous pairing between the F factor and the bacterial chromosome and a single crossover event between them is sufficient for the integration of the F factor into the bacterial chromosome (Fig. 18.3). The chromosome carrying an F plasmid is called an Hfr chromosome.
The Hfr factor has a stable attachment at a particular point in the host chromosome. The Hfr factor can break the bacterial chromosome and orient its transfer at different points to yield different Hfr strains. In strain H, the attachment point of Hfr factor is near threonine (thr), while in strain C, it is near lactose (lac) marker.
F Mediated Transfer of Bacterial Chromosome:
After effective contact and sex pili formation, the bacterial chromosome is ready for transfer. The transfer starts after 8 minutes of effective contact between the recipient and donor cells and it takes about 100 minutes for the complete transfer of the E. coli chromosome.
The entire bacterial chromosome can be mapped in 100 time units (1 unit = 1 minute), and the method is called conjugation mapping. Different Hfr strains initiate the transfer of their chromosomes at different points depending on the site of the F factor integration.
For example, the strain Hfr H transfers the gene for threonine synthesis (thr) first, strain Hfr J4 transfers the gene for thiamine synthesis (thi) first, the strain Hfr C transfers the gene for lactose (lac) first, while the strain Hfr G H transfers the gene xyl first. However, an F+ strain is not restricted to transfer genes from only a single point but it transfers it randomly.
In the Hfr chromosome, the integrated F-DNA is nicked at ori T to generate 5′ and 3′-ends. The nicked strand begins to separate from the 5′-end; a short sequence of the F-DNA first enters the recipient cell from its 5′-end, followed by the transfer of the donor bacterial chromosome.
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Thus one strand of the donor Hfr chromosome enters in the recipient cell from its 5′-end, while the 3′-end of this strand is utilized for rolling circle replication of the Hfr chromosome in the donor cell (Fig. 18.4). Simultaneously, the DNA strand being transferred into the F– recipient cell serves as a template for the synthesis of its complementary strand.
The process of transfer continues until the complete Hfr chromosome (including the integrated F factor) is transferred into or the mating is interrupted due to breaking of contacts between the conjugating bacteria. Recombination may occur between the recipient chromosome and the donor DNA.
Conjugation Mapping:
Jacob and Wollmann developed the interrupted mating technique for conjugation mapping. In this technique, the conjugating donor and recipient cells are separated at different time intervals, using a waring blender, and the genes transferred into the cell are determined by detecting the appearance of appropriate recombinants in the population.
It has been found that it takes 8 minutes for the start of the chromosome transfer, and that genes thr and leu were the first to be transferred from the Hfr H strain.
The time taken for the transfer of thr and leu was 8½ minute (including the initial 8 minutes). The gene azi entered the recipient cell after 9 minutes (8 + 1 minutes) and was placed at one unit distance on the map. Thus in Hfr H, the F factor is located after the methionine B locus (88 minutes) (Fig. 18.5). The functions of genetic markers shown in the Fig. 18.5, are given in Table 18.2.
F-Duction (Sex-Ductiori):
The F factor can be separated from the Hfr DNA to produce an independent F factor. In this process, the Hfr chromosome folds upon itself to form a figure “8” so that the F segment becomes aligned as a ring due to synapsis between the IS elements in the integration of the F factor into the host chromosome.
Now a crossing over within the paired IS elements leads to the formation of an independent F factor and a normal bacterial chromosome. But sometimes, synapsis of the IS element of the F factor may occur with a bacterial IS element other than that previously used for the integration.
In such situations, the separated F factors will contain a section of the bacterial DNA; such F factors are called “F prime” (F) or “substituted F factor” (Fig. 18.6). As a result, F’ factors can transmit bacterial genes from the F+ cells to the F– cells. For example, F carrying the lac+ gene will transmit it to an F– lac– cell; as a result, the latter will be converted into a lac+ cell. This process of gene transfer is called sexduction or F’ duction.
Interspecific Transfer of Plasmids:
Presence of similar genes on plasmids of distantly related bacteria indicates that interspecific transfer of plasmids can occur in many bacterial species. The sex factor or F plasmid of E. culi can be transferred to such different species as Shigella, Salmonella and Proteus. About 1/3 of freshly isolated E. coli strains have been found to contain conjugative plasmids. Such plasmids are found in about 30 bacterial genera most of which are Gram negative.
