Gene transfer in conjugation is one way only

The process of conjugation was initially envisaged as a fusion of the two partner cells to give a diploid zygote, which subsequently underwent meiosis to give haploid offspring with modified genotypes. The work of William Hayes, however, showed that the development of colonies of recombinant cells was dependant on the survival of only one of the participating strains, the other strain being required only as a donor of DNA.

It became apparent that in E. coli, there are two distinct mating types, which became known as F+ and F-, depending on whether or not they possessed a plasmid called the F (fertility) plasmid. This contains some 30 or 40 genes responsible for its own replication and for the synthesis of a thread-like structure expressed on the cell surface called a sex pilus. In a mixture of F+ and F- cells, the sex pilus contacts an F- cell, then

Cells of genotype a+ b

Cells of genotype a+ b

Figure 11.28 The Davis U-tube experiment. Two bacterial strains were placed in two arms of a U-tube, separated by a filter which did not permit the passage of cells. Although suction was used to transfer medium between compartments, no recombinants resulted. The results provided direct evidence that cell-to-cell contact is essential for conjugation to occur

Figure 11.28 The Davis U-tube experiment. Two bacterial strains were placed in two arms of a U-tube, separated by a filter which did not permit the passage of cells. Although suction was used to transfer medium between compartments, no recombinants resulted. The results provided direct evidence that cell-to-cell contact is essential for conjugation to occur contracts, to pull the two cells together. A single strand of plasmid DNA is then passed across a channel made between the two cells, and enters the F- cell (Figure 11.29). This then serves as a template for the production of the complementary second strand, and similarly the single strand left behind in the donor cell is replicated, leaving us with a double-stranded copy of the F plasmid in both cells. By acquiring the F plasmid, the recipient F- cell has been converted to F+.

When conjugation involves a form of donor cell called Hfr (high frequency of recombination), genes from the main bacterial chromosome may be transferred. In these, the F plasmid has become integrated into the main bacterial chromosome; and thus loses its ability to replicate independently (Figure 11.30). It behaves just like any other part of the chromosome, although of course it still carries the genes for conjugation and pilus formation. When conjugation occurs, a single strand of Hfr DNA is broken within the F sequence, and is transferred in a linear fashion, carrying behind it chromosomal DNA. Recombination in the recipient cell results in the replacement of a homologous segment of DNA by the transferred material, which is then faithfully replicated in subsequent generations. If transfer is uninterrupted, a process which takes around 100 minutes in E. coli, eventually the remaining stretch of F plasmid will enter the F- cell, bringing up the rear. The fragile nature of the pilus means, however, that transfer is rarely complete, and only a limited portion of the bacterial genome is transferred. This means that those F- cells receiving DNA from Hfr cells usually remain F-, unlike those in a cross with F+ cells, because the remainder of the F sequence is not transferred. It soon became clear that this phenomenon afforded a great opportunity to determine the relative positions of genes on the bacterial chromosome. This was done by interrupted mating experiments, in which the time allowed for conjugation is deliberately limited by mechanical breakage of the sex pili, and correlated with the phenotypic traits transferred to the recipient cells (Figure 11.31). By these means a genetic map of the bacterial chromosome could be developed.

One strand of the plasmid DNA in the F+ cell is nicked, and passes 5' end first into the conjugation tube.

Transfer continues into the F- cell.

= new strand

= new strand

The single strand in each cell acts as a template for the production of a second strand by the 'rolling circle' mechanism

Upon completion of replication, the cells separate. By gaining the F plasmid, the F-cell has been converted to F+.

Figure 11.29 Conjugation in bacteria. A single-stranded copy of the F plasmid passes across a conjugation tube from an F+ to an F- cell. Both this and the copy left behind act as templates for their own replication, leaving both cells with a complete F plasmid. This means that the recipient cell is converted from F- to F+.

F factor integrates into chromosome

Single strand of F episome nicked and passed to F-cell.

Chromosomal

Single strand of F episome nicked and passed to F-cell.

Chromosomal

Last part of F factor. Recipient cell only becomes Hfr if this is transferred.

F factor

F factor

Chromosomal gene transfer

Transferred chromosomal is replicated and may be incorporated into F-chromosome by recombination

DNA not incorporated is degraded

Transferred chromosomal is replicated and may be incorporated into F-chromosome by recombination

Figure 11.30 Conjugation with an Hfr cell results in transfer of chromosomal genes. Hfr cells are formed by the integration of the F factor into the bacterial chromosome. During conjugation, transfer begins part of the way along the F episome, and continues with chromosomal DNA. The amount of chromosomal material transferred depends on how long conjugation is able to proceed. Conjugation may be followed by recombination of transferred chromosomal material with its homologous sequence on the recipient cell's chromosome a

Percentage of recombinants with Hfr characters

Figure 11.31 In interrupted mating experiments, the conjugation tube is broken after different time periods. The time at which different genes are transferred reflects their relative positions on the bacterial chromosome. By plating out onto selective media, the order in which the different genes are transferred can be determined. The graph shows that the first genes to enter the F- cell are those present in the highest proportion of recombinants

Time (min)

Figure 11.31 In interrupted mating experiments, the conjugation tube is broken after different time periods. The time at which different genes are transferred reflects their relative positions on the bacterial chromosome. By plating out onto selective media, the order in which the different genes are transferred can be determined. The graph shows that the first genes to enter the F- cell are those present in the highest proportion of recombinants a

Box 11.9 It's that man again!

Not content with going down in history as the man who first demonstrated conjugation in bacteria, Joshua Lederberg was also, in 1952, one of the co-discoverers of transduction, along with Norton Zinder. Lederberg was to become a dominant figure in microbial genetics for over half a century. He was even responsible for coining the term 'plasmid'!

The integration of the F plasmid into the bacterial chromosome is reversible; thus Hfr cells can revert to F+. Excision of the integrated plasmid is not always precise, and sometimes a little chromosomal DNA is removed too. When this happens, the plasmid, and the cell containing it, are called F' ('F prime'); transfer of the plasmid to an F- cell takes with it the extra DNA from the host chromosome. The recipient genome thus becomes partially diploid (merodiploid), because it has its own copy, plus the 'guest' copy of certain genes.

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