In this article we will discuss about the presence of F factor in conjugation of bacteria.
Bacterial cells may carry besides the main chromosome, one or more small DNA molecules in the cytoplasm called plasmids. Of the various kinds of plasmids, a few are involved in conjugation and are called conjugative plasmids. One such conjugative plasmid is the sex element or fertility or F factor.
The presence or F factor in different strains has given rise to two mating types in bacteria namely, the donor which possesses the fertility factor and referred to as F+ strain, the second which lacks F factor is the F– strain. The F factor is itself the genetic element which is passed from donor to recipient cells during conjugation. There is no conjugation between two F+ strains or between two F– strains.
The F element contains about 2 per cent of the cell’s total DNA. It is capable of autonomous replication. It is made up of a circular, double stranded DNA molecule of molecular weight approximately 35 x 106. It contains about 15 genes, 8 of which control the formation of F-pili or sex pili which are hair-like appendages extending from the surface of F+ cells. F pili function in conjugation.
Structure of Pili:
The F pili originate from cell membrane and project outward beyond the cell wall (Fig. 17.2). The width of pili in different bacteria varies between 4 and 35 nm. The pilus is made up of a phosphate- carbohydrate-protein complex with a single polypeptide subunit called pilin of 11,000 to 12,000 Daltons.
Each pilin subunit has 2 residues of phosphate and one of glucose. The pili have been isolated and analysed by electron microscopy and X-ray diffraction techniques. The F pilus consists of a hollow cylinder 80 Å in diameter. The central hollow core is about 20 Å. The pilin subunits are arranged in the form of four helical chains.
The Mating Process of F Factor:
In a mixture of F+ and F– cells an F+ donor cell establishes contact with an F– recipient cell by the F pilus. The pilus is essential for recognition of recipient cell with which mating would take place.
After initial contact between the pilus and recipient cell is established, the pilus serves as a protoplasmic connection between the two cells and is called the conjugation tube. A donor cell devoid of pili cannot conjugate. The sex plasmid passes from the donor F+ to the recipient F– cell through the conjugation tube. Transfer takes place when DNA replicates by the rolling circle method (Fig. 17.3A).
A nick produced by an endonuclease in one strand of the plasmid DNA duplex produces a free 5′ and a 3′ end. The strand moves across the cytoplasmic bridge with the 5′ end first, into the F– cell. The second inner strand of the plasmid DNA duplex is retained in the F+ cell and synthesizes its complementary strand. The two cells separate after mating and are known as ex-conjugants. Thus the originally mixed population of F + and F– cells comes to have all F + cells only.
The transfer of sex element from F+ to F– cell has one more important feature. Not only can the plasmid exist in the cytoplasm as an autonomous entity, but it can also become incorporated in the main bacterial chromosome in a frequency of about 1 in 10,000 F+ cells.
Integration takes place at a specific site in the host chromosome which has homologous sequences. Such an integrated plasmid is known as episome and promotes the transfer of the main bacterial genophore from donor F+ to recipient F– cells during conjugation, an event followed by recombination.
High Frequency Recombination:
Sometime after the discovery of F+ strains, a special kind of strain was noticed which was several hundred times more fertile in crosses with F– than any known F+ strain. This strain was isolated by Lederberg et al. (1952) and was called Hfr or high frequency recombination. The Hfr strain produces about 1,000 times more prototrophs than in the F+ x F– cross.
In the mating system of Hfr strains the main bacterial chromosome containing an integrated F factor is transferred to F– cells. The Hfr bacteria arise spontaneously from F+ cells at a low frequency by integration of F factor in the main chromosome.
When Hfr cells are mixed with F– cells there is conjugation and a high frequency of transfer of only portions of the main bacterial chromosome (some selected markers) from donor to F– recipient cells. The recipient cell remains F–.
An F+ cell is converted to Hfr when F integrates into the main chromosome by reciprocal recombination. The process is reversible so that an Hfr cell becomes F+ when another recombinational event causes detachment of the F factor.
Hayes, Wollman and Jacob conducted experiments which demonstrated recombination during mating between Hfr and F–. They took wild type Hfr capable of synthesizing all its organic requirements, which could also utilise the sugars galactose and lactose, and was susceptible to being killed by streptomycin. The second strain they took was of the F– type which could not synthesise some amino acids (leucine and threonine), nor utilise galactose and lactose, and was resistant to streptomycin.
The Hfr and mutant F– strains were mixed and grown together. For analysing the progeny cells, samples of the cell mixture were grown on minimal medium containing streptomycin. Recombinants had appeared in the progeny.
Linear Chromosome Transfer by Hfr Strains:
Wollman and Jacob (1956) studied kinetics of genetic transfer by the interrupted mating technique. After mixing up two parental populations, cell samples are withdrawn at different intervals and agitated in a blender so that the mating pairs become separated and conjugation comes to an end. The mixture of cells is diluted and plated on selective medium and the number of recombinants formed during that interval are determined. The appearance of recombinants indicates formation of zygotes.
The different genetic markers are found to appear in the progeny of interrupted matings after different time periods have been allowed before mating is disrupted by agitation. Closely linked markers appear at the same time, whereas distantly placed markers appear at different times.
The markers threonine and leucine appear after about 8 minutes whereas gal appears after 26 minutes. The entire chromosome containing about 5 x 106 base pairs is transferred in 90 minutes. In this way it is possible to map locations of markers on the donor chromosome.
Further investigations showed that the Hfr donor cells transfer only a part of their genome to F– cells. Moreover, there are different Hfr strains which are distinct from each other in transferring a different part of the genome to F– cells. The E. coli genome is a closed circular loop.
In the Hfr donor cell the loop is broken at a point characteristic for that strain. The break occurs within the F element so that a part of it is at the leading end and is transferred to the F– cell, the other part of F is at the extreme distal end which trails behind. Transfer takes place by injection of the linear structure into the recipient cell.
The foremost or leading end carries with it gene loci nearest to it (Fig. 17.3B) until conjugation is interrupted. The transfer of DNA may be broken off at any time due to spontaneous rupture of the connection between conjugating cells.
After transfer to the F– cell the donor DNA fragment becomes incorporated into a homologous region of the host cell chromosome. The corresponding segment of the F– cell DNA is lost. Crossovers occur between the donor Hfr fragment and the F– host cell chromosome. This integration is essential before donor genetic markers can express themselves.
The presence of F factor in Hfr and F+ cells endows specific surface properties through formation of F pilus, due to which these cells can act as donors. In 1960 Loeb found out that certain bacteriophages could lyse only donor E. coli cells but not the recipient cells.
The phages R17 and M12 adsorb to pili present on donors but not on recipient cells and are referred to as male- specific phages. Further, the male-specific RNA phages are observed to adsorb along the length of the sex pilus, whereas male-specific DNA phages adsorb to the tip of the pilus.
The surface of the recipient cell also appears to play an important role in mating. When an F factor is present in a cell, it prevents the cell from acting as a recipient, so that super-infection does not occur. As this effect is due to a surface component which depends on the F factor, the phenomenon is known as surface exclusion.
Some of the surface proteins coded by the main bacterial chromosome genes are also involved in mating. The con– mutants of E. coli are not able to function as recipients and form mating pairs in conjugation. These mutants are found to be deficient in two of the surface proteins.
Although mating usually takes place between pairs of bacterial cells, Achtman (1975) found the presence of cell aggregates of 2-20 cells (called mating aggregates) which were involved in transferring DNA from donor to recipient cells.
The F’ Factor:
The F element can also become integrated into the main bacterial chromosome. Rarely an integrated F can undergo excision and become detached, carrying with it some bacterial genes that remain attached to it. Such an F element is called an F’ factor.
It behaves like the F factor of F+ cells and can be transferred to F– cells. Because F’ carries bacterial genes, it is able to pair with the corresponding region in the bacterial chromosome (Fig. 17.4). A bacterium receiving an F’ factor becomes a partial diploid for the bacterial genes carried by F’.
The Transfer Genes:
There are certain mutant strains in which transfer of F factor cannot take place. The transfer deficient mutants have been useful for identifying the presence of transfer (tra) genes in the F factor. Transfer genes are found to be necessary for conjugation. About 19 tra genes have been identified so far and these are classified into 4 groups.
Those in the first group control pilus formation and recognition of recipient cell. The second group genes are involved in stabilisation of mating pairs. Genes of the third group are required for some metabolic changes in DNA necessary for conjugation. There is only one gene in the fourth group (tra J) which controls the function of all the other tra genes.