Transfer of Chromosomal Genes Requires Plasmid Integration

Although many plasmids allow the cells carrying them to conjugate, usually only the plasmid itself is transferred through the conjugation bridge. Much less often, plasmids mediate transfer of the host chromosome when they move from one bacterial cell to another. In order to transfer chromosomal genes, a plasmid must first physically integrate itself into the chromosome of the bacterium. This event involves pairs of identical (or nearly identical) DNA sequences, one on the plasmid and the other on the chromosome. In practice, insertion sequences (see Ch. 15) are used for integration of the F-plasmid into the chromosome of E. coli (Fig. 18.14).

conjugation bridge Junction that forms between two cells and provides a channel for DNA to move from donor to recipient during conjugation fertility plasmid Plasmid that enables a cell to donate DNA by conjugation F-plasmid Fertility plasmid that allows E. coli to donate DNA by conjugation insertion sequence A simple transposon consisting only of inverted repeats surrounding a gene encoding transposase sex pilus Protein filament made by donor bacteria that binds to a suitable recipient and draws the two cells together Tra+ Transfer positive (refers to a plasmid capable of self-transfer) tra genes Genes needed for plasmid transfer

Transferable plasmids move from one cell to another via the conjugation bridge.

A single strand of newly made DNA is transferred from the donor to the recipient cell.

Plasmids unable to transfer themselves may be able to hitch-hike using the transfer systems of other plasmids.

Transferable plasmids sometimes move chromosomal DNA from one cell to another.

A) FORMATION OF MATING PAIRS

Chromosome

Transferable plasmid / Donor

Sex pilus

Chromosome

Sex pilus

Chromosome

B) FORMATION OF A CONJUGATION BRIDGE

FIGURE 18.11 Bacterial Conjugation

Certain plasmids, called Tra+ or transfer positive, are able to move a copy of their DNA into a different cell through a mechanism called bacterial conjugation. First the cell containing a Tra+ plasmid manufactures a rod-like extension on the surface of the outer membrane, called a sex pilus. The sex pilus binds to a nearby cell and pulls the two cells together. Once the cells are in contact, a connection is made between the two cells called the conjugation bridge. This connects the cytoplasm of the two cells, so the plasmid can transfer a copy of itself to the recipient cell.

FIGURE 18.12 Conjugating Cells of Escherichia coli

False-color transmission electron micrograph (TEM) of a male Escherichia coli bacterium (bottom right) conjugating with two females. This male has attached two F-pili to each of the females. The tiny bodies covering the F-pili are bacteriophage MS2, a virus that attacks only male bacteria and binds specifically to F-pili. Magnification: x11,250. Credit: Dr. L. Caro, Photo Researchers, Inc.

FIGURE 18.12 Conjugating Cells of Escherichia coli

False-color transmission electron micrograph (TEM) of a male Escherichia coli bacterium (bottom right) conjugating with two females. This male has attached two F-pili to each of the females. The tiny bodies covering the F-pili are bacteriophage MS2, a virus that attacks only male bacteria and binds specifically to F-pili. Magnification: x11,250. Credit: Dr. L. Caro, Photo Researchers, Inc.

A) REPLICATION

FIGURE 18.13 Plasmid Transfer Involving Rolling Circle Replication

A) During bacterial conjugation, the F-plasmid of E. coli is transferred to a new cell by rolling circle replication. First, one strand of the F-plasmid is nicked at the origin of transfer. The two strands start to separate and synthesis of a new strand starts at the origin (green strand). B) The single-strand of F-plasmid DNA that is displaced (pink strand) crosses the conjugation bridge and enters the recipient cell. The second strand of the F-plasmid is synthesized inside the recipient cell. Once the complete plasmid has been transferred, it is re-ligated to form a circle once again.

Origin of Single-strand r nick is made transfer

Origin of Single-strand r nick is made transfer

Double-stranded DNA

Single-strand enters recipient cell

Single-strand enters recipient cell

Synthesis of new DNA complementary to unbroken strand

Double-stranded DNA

B) TRANSFER

Synthesis of new DNA complementary to unbroken strand

Chromosome

Chromosome

Complementary strand synthesized in donor

Complementary strand synthesized in recipient

Complementary strand synthesized in donor

Complementary strand synthesized in recipient

FIGURE 18.14 Integration of F-Plasmid into Chromosome

If recombination occurs between two insertion sequences, one on the F-plasmid and one on the host bacterial chromosome, the entire F-plasmid is integrated into the chromosome.

FIGURE 18.14 Integration of F-Plasmid into Chromosome

If recombination occurs between two insertion sequences, one on the F-plasmid and one on the host bacterial chromosome, the entire F-plasmid is integrated into the chromosome.

FIGURE 18.15 Insertion Sequences on F-Plasmid and Chromosome

Insertion sequences are scattered throughout the F-plasmid and chromosome of E. coli. The F-plasmid has two IS3 elements and one IS2 element. Even more copies of IS2 and IS3 are found on the chromosome (not shown). A recombination event between any of the chromosomal IS2 or IS3 elements and the corresponding element on the F-plasmid will integrate the entire F-plasmid into the chromosome. Tn1000 (also known as gS) is another insertion sequence, although not generally involved in F-plasmid integration in E. coli.

Origin of vegetative replication

In order to mobilize chromosomal DNA, the plasmid must first integrate into the chromosome.

A variety of different insertion sequences are found on the chromosome of E. coli and in its plasmids and viruses. The F-plasmid, has three insertion sequences (Fig. 18.15); two copies of IS3 and a single copy of IS2. The chromosome of E. coli has 13 copies of IS2 and six copies of IS3 scattered around more or less at random. Integration of the F-plasmid may occur in either orientation at any of these 19 sites.

When an F-plasmid that is integrated into the chromosome transfers itself by conjugation, it drags along the chromosomal genes to which it is attached (Fig. 18.16). Just like the unintegrated F-plasmid, only a single strand of the DNA moves and the recipient cell has to make the complementary strand itself. Bacterial strains with an F-plasmid integrated into the chromosome are known as Hfr-strains because they transfer chromosomal genes at high frequency. A prolonged mating of 90 minutes or so is needed to transfer the whole chromosome of E. coli. More often, bacteria break off after a shorter period of, say, 15 to 30 minutes, and only part of the chromosome is transferred. Since different Hfr-strains have their F-plasmids inserted at different sites on the bacterial chromosome, transfer of chromosomal genes begins at different points. In addition, the F-plasmid may be inserted in either orientation. Consequently, gene transfer may be either clockwise or counterclockwise for any particular Hfr strain.

Hfr strains are useful for identifying the order of genes on the E. coli chromosome. In order to determine whether the recipient cell has received the gene in question, the donor and recipient strains must have different alleles of this gene that can be distin-

Hfr-strain Bacterial strain that transfers chromosomal genes at high frequency due to an integrated fertility plasmid

FIGURE 18.16 Transfer of Chromosomal Genes by F-Plasmid

An integrated F-plasmid can still induce bacterial conjugation and rolling circle transfer of DNA into another bacterial cell. Since rolling circle replication does not stop until the entire circle is replicated, the attached chromosome is also transferred into the recipient cell. First, a single-stranded nick is made at the or/T, or transfer origin of the integrated plasmid. The free 5' end (black triangle) enters the recipient cell through the conjugation bridge. Once inside the recipient, the second strand of DNA is synthesized. Notice that the transfer of the single stranded DNA does not end with the F-plasmid DNA and continues into the chromosomal DNA. Genes closest to the site of plasmid integration are transferred first (in the order a, b, c, d, e, f, in this example). The amount of chromosomal DNA that is transferred depends on how long the two bacteria remain attached by the conjugation bridge.

oriT origin of transfer

---Single-stranded nick is made at oriT

oriT origin of transfer

---Single-stranded nick is made at oriT

Single - strand is unrolled

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