8.6: Structure and Organization of Bacterial DNA... and Bacterial Sex

Sexual reproduction allows compatible genders (think male and female) to share genes, a strategy that increases species diversity. It turns out that bacteria and other single-celled organisms can also share genes and spread diversity with the help of plasmids. Plasmids are circular, extrachromosomal bits of DNA that contain their own genes (often, antibiotic-resistance genes), and that replicate themselves in bacteria. We’ll close this chapter with a look at sex (E. coli style!) and plasmid-assisted gene-mapping experiments showing linearly arranged genes on a circular bacterial DNA molecule (the bacterial “chromosome”).

E. coli sex begins when \(\rm F^{+}\) and \(\rm F^{-}\) cells meet. These “opposite” mating type cells can share DNA during conjugation. \(\rm F^{+}\) cells contain the extrachromosomal fertility plasmid, or F plasmid, that is separate from the E. coli chromosome. The F plasmid has genes that encode sex pili on \(\rm F^{+}\) cells, as well as factors needed to form a “mating bridge”, or conjugation tube. Figure 8.19 (below) illustrates the behavior of the F plasmid during conjugation.

Figure 8.19: Bacterial conjugation (sex in bacteria): F plasmids (Fertility plasmids) transfer chromosomal DNA from E. coli \(\rm F^{+}\) mating strain cells to \(\rm F^{-}\) strain cells, leading to genetic diversity.

When an \(\rm F^{+}\) (donor) cell meets an \(\rm F^{-}\) (recipient) cell, donor cell sex pili initiate recognition. A conjugation tube then forms, linking the cytoplasm of both cells. After the donor \(\rm F^{+}\) cell nicks one strand of the F plasmid DNA, the nicked strand enters the conjugation tube, rolling into the \(\rm F^{-}\) cell. Both the “traveling” DNA strand and the intact DNA circle remaining in the donor cell replicate (shown in red in Figure 8.19). E. coli conjugation can have different outcomes:

1. One of two semi-conservatively replicated F plasmids stays in the donor cell, and the other ends up in the recipient cell, making the recipient cell a new \(\rm F^{+}\) donor cell!
2. The F plasmid inserts itself into recipient cell’s chromosomal DNA, typically at a specific site in the DNA where there is sufficient sequence similarity between the plasmid and the chromosomal DNA. This allows insertion by recombination. With the fertility-factor genes now part of its chromosome, the recipient cell becomes an Hfr (High-frequency recombination) cell, and it will produce Hfr-strain progeny cells.

In both outcomes the mating type of the recipient \(\rm F^{-}\) cell becomes either an \(\rm F^{+}\) or an Hfr cell that can initiate mating with other \(\rm F^{-}\) cells as illustrated in Figure 8.20.

Figure 8.20: After conjugal F plasmid transfer, \(\rm F^{-}\) recipients can become \(\rm F^{+}\) or, if the plasmid integrates into the recipient genome, it will become an Hfr cell.

Hfr cells readily express their integrated F-plasmid genes, and like F+ cells, they develop sex pili and form a conjugation tube with an \(\rm F^{-}\) cell. One strand of the bacterial chromosomal DNA will be nicked at the original insertion site of the F plasmid. The next events parallel the replicative transfer of an F plasmid during \(\rm F^{+}\)/\(\rm F^{-}\) conjugation, except that only part of the Hfr donor chromosomal DNA is transferred, as seen in Figure 8.21.

Figure 8.21: During conjugation with Hfr-donor strain cells, a portion of the donor’s chromosomal DNA follows the excised plasmid across the conjugation bridge into the \(\rm F^{-}\) recipient cell.

In the illustration, the F plasmid (red) has inserted itself in front of an A gene so that when it enters the conjugation tube, it brings along several E. coli chromosomal genes. Because of the size of the bacterial chromosome, only a few bacterial genes enter the recipient gene before transfer is aborted. But in the brief time of DNA transfer, at least some genes did get into the recipient \(\rm F^{-}\) strain where they can be expressed. Here is the outline of an experiment that allowed bacterial genes on a circular DNA chromosome to be mapped:

1. Hfr cells containing functional A, B, C, and D genes were mated with recipient cells containing mutants of the A, B, C, or D genes.
2. Conjugation was mechanically disrupted at different times after the formation of a conjugation tube.
3. Recipient cells from each of the disrupted conjugations were then grown into culture and analyzed for specific gene function.

In this hypothetical example, the results were that a recipient cell with a mutant A gene acquired a wild-type A gene (and therefore A-gene function) when there was only a short time before conjugation disruption. Progressively longer conjugation times (measured in separate experiments) were required to transfer genes B, then C, then D to the recipient cell. Thus, the order of these genes on the bacterial chromosome was -A-B-C-D-.

The timing of conjugation that led to \(\rm F^{-}\) mutants acquiring a functional gene from the Hfr strain was so refined that it was possible to determine not only the gene locus but also the size (length) of the genes! Thus, the time to transfer a complete gene to an \(\rm F^{-}\) cell reflects the length of the gene, and also, the linear order of genes on bacterial DNA.

Recall that genes mapped along the eukaryotic chromosomes had already implied a linear order of the genes. However, little was known about eukaryotic chromosome structure at the time, and the role of DNA as the “stuff of genes” was not yet appreciated. These bacterial mating experiments demonstrated for the first time that genes are linearly arranged not just along a chromosome but also along the DNA molecule.

Over time, many bacterial genes were mapped all along the E. coli chromosome by isolating many different Hfr strains in which an F plasmid had been inserted into different sites around the DNA circle. These Hfr strains were mated to \(\rm F^{-}\) bacteria, each with mutations in one or another known bacterial gene. As in the original “ABCD” experiment, the order of many genes was determined and even shown to be linked at a greater or lesser distance to those ABCD genes and each other. Using these Hfr strains in conjugation experiments, it was shown that in fact, the different Hfr cells transferred different genes into the recipient cells in the order implied by the chromosome map illustrated in Figure 8.22

Figure 8.22: Map of the E. coli chromosome (its genome!) based on conjugation of \(\rm F^{-}\) recipient cells with different strains of Hfr donor cells (indicated as Hfr 1, 2, 3…): the different Hfr donors are cells in from which Hfr plasmids have integrated at different places on a recipient cell’s chromosome. When the Hfr strains transfer DNA to new \(\rm F^{-}\) cells, they bring along different reg

What’s more, when the experiment was done with Hfr4 (in this generic diagram), the order of genes transferred after longer times of conjugation was found to be -V-W-X-Y-Z-A-B-…. The obvious conclusion is that an E. coli DNA molecule (i.e., its “chromosome”) is a closed circle!

We will see visual evidence of circular E. coli chromosomes in the next chapter, with some discussion of how this evidence informed our understanding of DNA replication.