Conjugation

Another important and common mechanism of gene transfer in both Gram-positive and Gram-negative bacteria is conjugation. The process is quite different in the two groups and we will only consider the congugation in Gram-negative bacteria. Conjugation, in contrast to transformation and transduction, requires contact between donor and recipient cells. This requirement can be shown through the following experiment. If two different auxotrophic mutants are placed on either side of a filter through which fluids, but not bacteria, can pass, recombination does not occur. If the filter is removed, however, allowing cell-to-cell contact, recombination takes place. Some cells in the population transfer plasmids whereas other cells can transfer their chromosome. Conjugation is a complex process and many aspects are not understood even though it was first observed in E. coli more than 50 years ago. ■ plasmid, p. 66

Plasmid Transfer

Populations of E. coli can be divided into two types of cells. One, the donor cell, contains an F or fertility plasmid and is designated F+. The other, the recipient cell, does not contain this plasmid and is called FDNA is transferred only in one direction, from F+ to F:—that is, in a polar fashion. Consequently, F + cells are often referred to as males and the F: cells as females. The F plasmid codes for the synthesis of a structure, the sex or F pilus, the protein appendage that attaches the donor to the recipient cell (figure 8.17). The plasmid also carries information required for its own transfer. Donor cells can

Pili For Conjugation

2 mm

Figure 8.17 Sex or F Pilus Holding Together Donor and Recipient Cells of E. co/i During DNA Transfer The tiny knobs on the pilus are bacterial viruses that have adsorbed to the pilus.

2 mm

Figure 8.17 Sex or F Pilus Holding Together Donor and Recipient Cells of E. co/i During DNA Transfer The tiny knobs on the pilus are bacterial viruses that have adsorbed to the pilus.

transfer their F plasmid but not their chromosome into recipient cells. ■ F pilus, p. 65

Plasmid transfer can be divided into four steps (figure 8.18).

Step 1: Contact between donor and recipient cells. The sex pili of the donor cells recognize and bind to specific receptor sites on the cell walls of the recipient cells. When donor and recipient cells are mixed together, the sex pili likely act as grappling hooks, pulling the two cells together. Step 2: Mobilization or activation of DNA transfer. The plasmid becomes mobilized for transfer when a plasmid-encoded enzyme cleaves one strand of the plasmid at a specific nucleotide sequence, termed the origin of transfer. This results in the formation of a single-stranded DNA molecule with a free end.

Chromosome F plasmid

Sex pilus

Chromosome F plasmid

Sex pilus

Conjugation Between Prime

The sex pilus contacts the recipient F- cell.

The plasmid is activated for transfer when an endonuclease cleaves one strand of DNA at the origin of transfer.

The sex pilus retracts and pulls the donor and recipient cells together. The F plasmid is transferred as a single-stranded DNA molecule.

The complementary strands to both F plasmid strands are synthesized in the donor and recipient cells. Both cells are F+ and synthesize the sex pilus.

F+ cell

F+ cell

Figure 8.18 Conjugation—Transfer of the F Plasmid The exact process by which the donor DNA passes to the recipient cells is not known.

8.9 Conjugation 207

Step 3: Plasmid transfer. Within minutes of the F+ cell contacting the F: cell, a single strand of the F plasmid, beginning at the origin of transfer, enters the F: cell. This transfer takes about 2 minutes. There is no evidence that the DNA passes through the pilus. Note that a single strand of the F plasmid remains in the donor cell.

Step 4: Synthesis of a functional plasmid inside the recipient and donor cells. Once inside the recipient cell, a complementary strand to the single-stranded transferred DNA is synthesized. Likewise, a strand complementary to the single-stranded plasmid DNA remaining in the donor is synthesized. Thus, both the donor and recipient cells contain a copy of the F plasmid and are therefore F+. Both cells can act as donors of the F plasmid. This explains why a single F+ cell of E. coli mixed with a population of F: cells can convert the entire population to F+ after overnight growth of the culture. Cells can spontaneously lose their plasmids, which explains why not all cells are F+. Cells that have lost their plasmid are said to be cured.

Transfer of Plasmids Other than F

Plasmids other than F are also transferred by conjugation. The F plasmid is termed a self-transmissible plasmid because it carries all the genetic information necessary for its own transfer. The plasmid codes for two functions; the synthesis of the sex pilus, and the mobilization of its DNA for transfer. Other plasmids are not self-transmissible— they do not code for both functions. A self-transmissible plasmid, however, can often provide the missing function to a non-self-transmissible plasmid if both are in the same cell. Thus, a non-self-transmissible plasmid in the same cell as the F plasmid will often be transferred.

The F+ donor cell containing an F plasmid is capable of synthesizing a sex pilus.

The sex pilus contacts the recipient F- cell.

The plasmid is activated for transfer when an endonuclease cleaves one strand of DNA at the origin of transfer.

The sex pilus retracts and pulls the donor and recipient cells together. The F plasmid is transferred as a single-stranded DNA molecule.

The complementary strands to both F plasmid strands are synthesized in the donor and recipient cells. Both cells are F+ and synthesize the sex pilus.

F+ cell

F+ cell

Figure 8.18 Conjugation—Transfer of the F Plasmid The exact process by which the donor DNA passes to the recipient cells is not known.

Chromosome Transfer

Thus far, we have discussed only the transfer of plasmids by conjugation. However, the bacterial chromosome is sometimes transferred to F: cells by a few cells in the F+ population. These donor cells are not in fact F+ but arise from F+ cells as a result of the F plasmid becoming integrated on rare occasions, into the donor cell chromosome at specific sites (figure 8.19). This integration occurs by recombination at sites on the chromosome that are homologous to sites on the plasmid. These sites are frequently insertion sequences (ISs), a type of trans-posable element which will be discussed later in this chapter. The chromosome contains many copies of several different ISs. The cells able to transfer the chromosome

208 Chapter 8 Bacterial Genetics

E.coti chromosome

F plasmid

F plasmid

Integrated Plasmid

Hfr CELL

Figure 8.19 Hfr Formation Integration of the F plasmid into the bacterial chromosome to form Hfr.There are homologous sites, insertion sequences, on the F plasmid and the chromosome that allow the integration to occur.There is no replacement of DNA in the chromosome; the F plasmid increases the size of the chromosome.

are called Hfr for high frequency of recombination. In the Hfr cell, the F plasmid replicates as part of the chromosome. ■ insertion sequence, p. 213

Many features of chromosome transfer are similar to those involved in plasmid transfer (figure 8.20). These similarities include the following:

■ The Hfr cell contacts the F: cell by means of the sex pilus.

■ DNA is transferred as a single strand and is made double stranded in the recipient cell.

■ Donor cells remain Hfr, just as F+ donor cells remain F+.

■ Transfer is very efficient, and all of the donor cells can transfer DNA.

There are, however, two major differences in the transfer of the chromosome compared to plasmid transfer.

First, most recipient cells that receive chromosomal DNA remain F:, in contrast to recipient cells conjugating with F+ cells. Consequently, a recipient cell receiving chromosomal DNA cannot transfer the chromosomal DNA to

Figure 8.20 Conjugation—Transfer of Chromosomal DNA The DNA is transferred as a single-stranded DNA molecule.The recipient genes are designated with a prime („ ).The corresponding forms in the donor lack the prime („).

Donor Cell F+

F plasmid

Recipient Cell F-

Donor Cell F+

F plasmid

Recipient Cell F-

Figure 8.20 Conjugation—Transfer of Chromosomal DNA The DNA is transferred as a single-stranded DNA molecule.The recipient genes are designated with a prime („ ).The corresponding forms in the donor lack the prime („).

Conjugation

The sex pilus contacts the recipient F- cell and pulls the cells together. Genes are indicated as 1, 2, 3, and 4.

The donor DNA transferred is integrated into the recipient cell's chromosome. The recipient cell remains F-. The donor cell synthesizes a strand complementary to the untransferred strand of DNA and remains Hfr.

Recipient ' cell DNA

Integration of donor chromosomal DNA

The donor cell synthesizes a sex pilus.

The sex pilus contacts the recipient F- cell and pulls the cells together. Genes are indicated as 1, 2, 3, and 4.

The donor chromosome is transferred as single- stranded DNA starting at the origin of transfer. Gene 1, closest to the origin, is transferred first. The integrated F plasmid is both at the beginning and at the end of the DNA being transferred.

The donor and recipient cells separate, usually before the entire chromosome is transferred. The single strand in both the recipient cell and donor cell are made double-stranded.

The donor DNA transferred is integrated into the recipient cell's chromosome. The recipient cell remains F-. The donor cell synthesizes a strand complementary to the untransferred strand of DNA and remains Hfr.

Recipient ' cell DNA

Integration of donor chromosomal DNA

Hfr Cell

F- Cell

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Perspective, Fourth Edition

8.10 Plasmids 209

other cells in the population. The transfer of the chromosome starts at the origin of transfer in such a way that a piece of the F plasmid integrated into the donor chromosome is transferred first (see figure 8.20). The rest of the F plasmid is at the other end of the chromosome, however, so that the entire chromosome must be transferred if the recipient cell is to become Hfr. The two cells must remain in close contact for transfer to occur, and they rarely remain in contact for the length of time required for the transfer of the entire donor chromosome (100 minutes). Therefore, only genes near the origin of transfer are transferred at a high frequency, and the recipient cells usually remain F:. The origin of transfer varies in its location on the chromosome depending on the site of integration of the F+ plasmid.

Second, once inside the recipient cell, the donor DNA integrates into the recipient chromosome by replacing homologous genes in the recipient cell through recombination (see figure 8.20). The fragment of the chromosome does not contain an origin of replication. Unless it integrates into the F: chromosome, it cannot replicate and will be destroyed. This contrasts with the transfer of plasmids, which replicate independently of the chromosome and therefore do not need to integrate into the recipient cell chromosome.

F' Donors

The F plasmid in the Hfr strain can be excised from the chromosome; thus, the process of F plasmid incorporation into the chromosome is reversible. In some instances in the process of excision, an error occurs and a small piece of the bacterial chromosome remains attached to the F plasmid (figure 8.21). This F plasmid with its piece of attached chromosome is called F' (F prime), and like the F plasmid it is rapidly and efficiently transferred to all F: cells in the population. Consequently, any chromosomal genes attached to the F plasmid are also transferred to all cells. The F' plasmid usually remains extrachromosomal—that is, it does not become a part of the recipient cell's chromosome. On rare occasions, however, it can become incorporated into the chromosome of recipient cells, which then become Hfr since they contain the F plasmid integrated into the chromosome.

The three mechanisms of DNA transfer are compared in table 8.3.

FORMATION OF F' CELL

Hfr Cell

Integrated F plasmid

F' Cell

F' plasmid

Hfr Cell

Integrated F plasmid

Chromosome

Chromosomal DNA

Chromosome

Figure 8.21 Formation of F' Plasmid This F' plasmid has the transfer properties of the F plasmid but carries chromosomal DNA.This process is reversible.

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Responses

  • natalia murray
    Is the integrated f plasma into chromosomes is reversible?
    3 years ago
  • TEKLE
    Does the hfr cell remain the same after conjugation?
    1 year ago

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