In 1946, Joshua Lederberg and Edward Tatum demonstrated that bacteria can transfer and recombine genetic information, paving the way for the use of bacteria in genetic studies. In the course of their research, Lederberg and Tatum studied auxotrophic strains of E. coli. The Y10 strain required the amino acids threonine (and was genotypically thr⁻) and leucine (leu⁻) and the vitamin thiamine (thi⁻) for growth, but did not require the vitamin biotin (bio⁺) or the amino acids phenylalanine (phe⁺) and cysteine (cys⁺); the genotype of this strain can be written as thr⁻ leu⁻ thi⁻ bio⁺ phe⁺ cys⁺. The Y24 strain had the opposite set of alleles: it required biotin, phenylalanine, and cysteine in its medium, but it did not require threonine, leucine, or thiamine; its genotype was thr⁺ leu⁺ thi⁺ bio⁻ phe⁻ cys⁻. In one experiment, Lederberg and Tatum mixed Y10 and Y24 bacteria together and plated them on minimal medium (Figure 7.8). Each strain was also plated separately on minimal medium.

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Alone, neither Y10 nor Y24 grew on minimal medium: each strain required nutrients that were absent. Strain Y10 was unable to grow because it required threonine, leucine, and thiamine, which were absent in the minimal medium; strain Y24 was unable to grow because it required biotin, phenylalanine, and cysteine, which also were absent from the minimal medium. When Lederberg and Tatum mixed the two strains, however, a few colonies did grow on the minimal medium. These prototrophic bacteria must have had genotype thr⁺ leu⁺ thi⁺ bio⁺ phe⁺ cys⁺. Where had they come from?

If mutations were responsible for the prototrophic colonies, then some colonies should also have grown on the plates containing Y10 or Y24 alone, but no bacteria grew on those plates. Multiple simultaneous mutations (thr⁻ → thr⁺, leu⁻ → leu⁺, and thi⁻ → thi⁺ in strain Y10 or bio⁻ → bio⁺, phe⁻ → phe⁺, and cys⁻ → cys⁺ in strain Y24) would have been required for either strain to become prototrophic by mutation, which was very improbable. Lederberg and Tatum concluded that some type of genetic transfer and recombination had taken place:

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What they did not know was how it had taken place.

To study this problem, Bernard Davis constructed a U-shaped tube (Figure 7.9) that was divided into two compartments by a filter with fine pores. This filter allowed liquid medium to pass from one side of the tube to the other, but the pores of the filter were too small to allow the passage of bacteria. Two auxotrophic strains of bacteria were placed on opposite sides of the filter, and suction was applied alternately to the ends of the U-tube, causing the medium to flow back and forth between the two compartments. Despite hours of incubation in the U-tube, bacteria plated on minimal medium did not grow; there had been no genetic exchange between the strains. The exchange of bacterial genes clearly required direct contact, or conjugation, between the bacterial cells.

F⁺ AND F⁻ CELLS In most bacteria, conjugation depends on a fertility (F) factor that is present in the donor cell and absent in the recipient cell. Cells that contain the F factor are referred to as F⁺, and cells lacking the F factor are F⁻.

The F factor is an episome that contains an origin of replication and a number of genes required for conjugation (see Figure 7.6). For example, some of these genes encode sex pili (singular, pilus), slender extensions of the cell membrane. A cell containing the F factor produces sex pili, one of which makes contact with a receptor on an F⁻ cell (Figure 7.10) and pulls the two cells together. DNA is then transferred from the F⁺ cell to the F⁻ cell. Conjugation can take place only between a cell that possesses the F factor and a cell that lacks the F factor.

In most cases, the only genes transferred during conjugation between an F⁺ and F⁻ cell are those on the F factor (Figure 7.11a and b). Transfer is initiated when one of the DNA strands on the F factor is nicked at an origin of transfer (oriT). One end of the nicked DNA separates from the circular F factor and passes into the recipient cell (Figure 7.11c). Replication takes place on the nicked strand, proceeding around the F factor in the F⁺ cell and replacing the transferred strand (Figure 7.11d). Because the F factor in the donor cell is always nicked at the oriT site, this site always enters the recipient cell first, followed by the rest of the plasmid. Thus, the transfer of genetic material has a defined direction. Inside the recipient cell, the single strand is replicated, producing a circular, double-stranded copy of the F plasmid (Figure 7.11e). If the entire F factor is transferred to the recipient F⁻ cell, that cell becomes an F⁺ cell.

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Hfr CELLS Conjugation transfers genetic material in the F plasmid from F⁺ to F⁻ cells, but it does not account for the transfer of chromosomal genes observed by Lederberg and Tatum. In Hfr (high-frequency recombination) bacterial strains, the F factor is integrated into the bacterial chromosome (Figure 7.12). Hfr cells behave like F⁺ cells, forming sex pili and undergoing conjugation with F⁻ cells.

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In conjugation between Hfr and F⁻ cells (Figure 7.13a), the integrated F factor is nicked, and the end of the nicked strand moves into the F⁻ cell (Figure 7.13b), just as it does in conjugation between F⁺ and F⁻ cells. But because, in an Hfr cell, the F factor has been integrated into the bacterial chromosome, the chromosome follows the F factor into the recipient cell. How much of the bacterial chromosome is transferred depends on the length of time that the two cells remain in conjugation.

Inside the recipient cell, the donor DNA strand is replicated (Figure 7.13c), and crossing over between it and the original chromosome of the F⁻ cell (Figure 7.13d) may take place. This chromosomal gene transfer between Hfr and F⁻ cells explains how the recombinant prototrophic cells observed by Lederberg and Tatum were produced. After crossing over has taken place in the recipient cell, the donated chromosome is degraded, and the recombinant recipient chromosome remains (Figure 7.13e), to be replicated and passed on to later generations of bacterial cells by binary fission (cell division).

In a mating of Hfr × F⁻, the F⁻ cell almost never becomes F⁺ or Hfr because the F factor is nicked in the middle in the initiation of strand transfer, which places part of the F factor at the beginning and part at the end of the strand that is transferred. To become F⁺ or Hfr, the recipient cell must receive the entire F factor, which requires that the entire donor chromosome be transferred. This event happens rarely because most conjugating cells break apart before the entire chromosome has been transferred.

The F plasmid in F⁺ cells integrates into the bacterial chromosome, causing an F⁺ cell to become Hfr, at a frequency of only about 1 in 10,000. This low frequency accounts for the low rate of recombination observed by Lederberg and Tatum in their F⁺ cells. The F factor is excised from the bacterial chromosome at a similarly low rate, causing a few Hfr cells to become F⁺.

F′ CELLS When an F factor is excised from the bacterial chromosome, a small amount of the bacterial chromosome may be removed with it, and these chromosomal genes will then be carried with the F plasmid (Figure 7.14). Cells containing an F plasmid with some bacterial genes are called F prime (F′) cells. For example, if an F factor integrates into a chromosome adjacent to the lac genes (genes that enable a cell to metabolize the sugar lactose), the F factor may pick up lac genes when it is excised, becoming an F′ lac cell. F′ cells can conjugate with F⁻ cells because F′ cells possess the F plasmid with all the genetic information necessary for conjugation and gene transfer. Characteristics of E. coli cells of different F types (called mating types) are summarized in Table 7.2.

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During conjugation between an F′ cell and an F⁻cell, the F plasmid is transferred to the F⁻ cell, which means that any genes on the F plasmid, including those from the bacterial chromosome, may be transferred to F⁻ recipient cells. This process produces partial diploids, or merozygotes, which are cells with two copies of some genes, one on the bacterial chromosome and one on the newly introduced F plasmid. The outcomes of conjugation between different mating types of E. coli are summarized in Table 7.3.

MAPPING BACTERIAL GENES WITH INTERRUPTED CONJUGATION The transfer of DNA that takes place during conjugation between Hfr and F⁻ cells allows bacterial genes to be mapped. In conjugation, as we have seen, the chromosome of the Hfr cell is transferred to the F⁻ cell. Transfer of the entire E. coli chromosome requires about 100 minutes; if conjugation is interrupted before 100 minutes have elapsed, only part of the donor chromosome will pass into the F⁻ cell and have an opportunity to recombine with the recipient chromosome. Chromosome transfer always begins within the integrated F factor and proceeds in a continuous direction, so genes are transferred according to their sequence on the chromosome. This knowledge can be used to map bacterial genes by mixing Hfr and F⁻ cells that differ in genotype and interrupting conjugation at regular intervals. The times required for individual genes to be transferred indicate their relative positions on the chromosome. In most genetic maps, distances are expressed as recombination frequencies; however, in bacterial gene maps constructed with interrupted conjugation, the basic unit of distance is a minute. View Animation 7.1 to see how genes are mapped using interrupted conjugation.

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