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Modern Genetic Analysis Chapter 9. The Genetics of Bacteria and Phages
Bacterial Conjugation

Conjugation is merely the fusion of two compatible bacterial cells. Bringing two genotypes together and allowing them to conjugate is the equivalent of making a cross in eukaryotes. Our discussion of conjugation will center on the gut bacterium Escherichia coli (E. coli). Conjugation and gene transfer in E. coli are driven by a circular DNA plasmid called the fertility factor or sex factor (F), which is found in some but not all cells. Hence to understand how to make a cross in E. coli, we have to understand the properties of F.

The Remarkable Properties of the F Plasmid

Cells carrying the F plasmid are designated F+, and those lacking it are F. The F plasmid contains approximately 100 genes, which give the plasmid several important properties:

1. The F plasmid can replicate its own DNA, allowing the plasmid to be maintained in a dividing cell population (Figure 9-3a).
2. Cells carrying the F plasmid promote the synthesis of pili (singular, pilus) on the bacterial cell surface. Pili are minute proteinaceous tubules that allow the F+ cells to attach to other cells and maintain contact with them; that is, to conjugate (Figure 9-3b).
3. F+ and F cells can conjugate. When conjugation occurs, the F+ cells can act as F donors. The F plasmid DNA replicates and the newly synthesized copy of the circular F molecule is transferred to the F recipient (Figure 9-3c). However, a copy of F always remains behind in the donor cell. The recipient cell becomes converted into F+, because it now contains a circular F genome. The transfer of the F plasmid from F+ to F is rapid, so the F plasmid can spread like wildfire throughout a population from strain to strain.
4. F+ cells are usually inhibited from making contact with other F+ cells; therefore the F plasmid is not transferred from F+ to F+.
5. Sometimes F carries within its genome one or more IS (insertion-sequence) elements (see Chapter 13). An IS element is a mobile segment of DNA that moves from place to place within the host chromosome or between chromosome and plasmid. The existence of a specific IS element both in the plasmid and in the chromosome affords a site at which homologous crossing-over occasionally occurs. A crossover between the two circular DNAs leads to the integration of the plasmid into the bacterial chromosome, as shown in Figures 9-4 below and 9-5a on the following page. When this integration occurs, F can drive the transfer of the entire host chromosome into the recipient cell, along with its own integrated F DNA (Figure 9-5b).

This last process, the associated transfer of F and host genes, has some interesting features. First, in any population of cells containing the F factor, F will integrate into the chromosome only in a small fraction of cells (Figure 9-5c). These few cells can now transfer chromosomal alleles to a second strain. The transfer is detectable because donor and recipient alleles recombine to produce genetic recombinants that can be identified. Indeed, the observation of recombinants led to the initial discovery of gene transfer by conjugation (see Genetics in Process 9-1 on page 277).

It is possible to isolate the rare cells in which the F factor is integrated into the host chromosome from the bacterial population and to cultivate pure strains derived from these cells. In such strains, every cell donates chromosomal alleles during F transfer, so the frequency of recombinants for these strains is much higher than it is for cells in the original population, where the F factor is not integrated in most cells. Therefore, strains with an integrated F factor are termed high frequency of recombination (Hfr) strains to distinguish them from normal F+ strains, which contain only a few rare Hfr cells and thus display only a low frequency of recombination for the strain as a whole. Because they transfer chromosomal markers efficiently, Hfr strains are the ones used for genetic mapping, as we shall see later on.

6. The integrated F factor occasionally leaves the chromosome of an Hfr cell and moves back to the cytoplasm, in some rare cases carrying a few host chromosomal genes along with it (Figure 9-5d). This modified F, called F (pronounced "F prime"), can now transfer these specific host genes to a recipient (F) cell in an infectious manner, in the same way that F is spread. Thus, the recipient cell now contains two copies of the same geneone resident copy on its bacterial chromosome and one copy on the newly transferred cytoplasmic F factor.
Recombination between Donor and Recipient DNA

Conjugation allows genes from two different parental cells to come together in the same cell and hence provides an opportunity for recombination to occur. Hence mapping analysis is possible.

All conjugations ("crosses") are by definition of the type Hfr (donor) × F (recipient). After cell union, the Hfr chromosome replicates in a peculiar manner that reels out a single-stranded DNA molecule, which is then transferred linearly into the F cell. The replication and transfer begin at a specific point at one side of the integrated F, called the origin (O). Genes close to the origin are transferred first. The integrated F factor would be transferred last; however, in most conjugations, the chromosomal transfer process stops before F enters (Figure 9-6).

Once inside the F cell, the linear single-stranded DNA molecule acts as a polymerization template and is converted into a DNA double helix. This linear donor fragment is the exogenote, and the resident F chromosome is the endogenote. As a free molecule, the exogenote cannot replicate and will become lost, but because exogenotes and endogenotes are homologous, crossing-over can take place between them. A single crossover between a linear molecule (the exogenote) and a circular one (the endogenote) would produce a single long molecule that would be inviable. However, two crossovers would integrate a part of the donor genome into the recipient. It is in this way that recombination takes place (Figure 9-6). (Note that, although such integrative exchanges can be considered to be double crossovers in the formal genetic sense, at the DNA level the mechanism is a single integration event in which a long donor segment replaces the equivalent segment in the recipient.)

Gene transfer and recombination provide the key to mapping the bacterial chromosome. There are two main methods: mapping by interrupted conjugation, which produces a low-resolution map of large parts of the genome, and mapping by recombinant frequency, which produces a higher-resolution map of a smaller region.

Mapping by Interrupted Conjugation

In mapping by interrupted conjugation, the Hfr and F cells are mixed, and conjugation proceeds. Then, at fixed times, the F cells are sampled to determine which donor alleles have entered. This sampling is accomplished by using a kitchen blender to separate the joined cells, resulting in interrupted conjugation. After separation, the Hfr cells are selectively killed, and the remaining F cells, the exconjugants, are tested to see which of the donor alleles have entered and stably recombined with the endogenote. The times at which various donor alleles first appear in the exconjugants are calculated. If a donor allele a+ enters the recipient at 5 minutes after union and allele b+ enters at 8 minutes, then the two genes are said to be 3 minutes apart on the chromosome. The map units in this case are minutes. Like the maps based on crossover frequencies, these linkage maps are purely genetic constructions. Although the amount of DNA corresponding to a minute is now known, when the method was first devised this was not the case.

Let's analyze a typical cross in which the order and map position of the genes under study are not known. In this particular cross, the genes by which the parents differ will be azi (resistance or sensitivity to sodium azide), gal (ability or inability to utilize galactose as an energy source), lac (ability or inability to utilize lactose as an energy source), and ton (resistance or sensitivity to bacteriophage T1). A streptomycin-sensitivity allele (strs) in the Hfr and a streptomycin-resistance allele (strr) in the recipient are used to selectively kill the Hfr cells after conjugation. Selective killing is accomplished by adding streptomycin to the mixture of cells after interrupting the conjugation. It is advantageous if such an Hfr "contraselecting" allele enters close to last, because then it will only rarely enter the F; in other words, it should be close to the integrated F factor. Hence the position of the contraselected gene must have been established in previous experiments. The parents of the cross under consideration here are as follows, where the unmapped genes are written in alphabetical order:

The results of the interrupted-mating experiment are shown in Figure 9-7. The azir gene is the first to be detected, entering at 8 minutes, followed by tonr, lac+, and gal+ in that order. Therefore not only is gene order on the chromosome map established, but map distances in minutes also are obtained, as shown in Figure 9-8.

Note, from Figure 9-7, that alleles transferred early are found in a high percentage of F exconjugants, but the late alleles are found in only a small proportion. The reason for this difference is either that transfer spontaneously stops or that the chromosome breaks. However, this result does not affect the time-of-entry calculations.

The relative positions of the azi, ton, lac, and gal genes were established in our experiment. However, the chromosomal region containing these loci might be only a small proportion of the entire chromosome. The complete map is obtained from many such interrupted conjugation experiments, in which parental strains heteroallelic for different combinations of genes are used; then the overall map is pieced together from the complete set of data. In Hfrs of different origin, the integrated F factor can be in different positions and different orientations. Examples of the positions and orientations of F in different Hfrs are shown in Figure 9-9.

High-Resolution Mapping by Recombinant Frequency

Interrupted-mating experiments provide a rough set of gene locations over the entire map. As we learned, the genes are mapped by time of entry. In such experiments, the exogenote must integrate by a double recombination event, but the mapping method is not based on any measurement of recombinant frequencies. However, to provide a higher-resolution method for measuring the sizes of smaller map distances, recombinant frequencies are used.

Suppose that we undertake an experiment to map three genesmet, arg, and leuby recombinant frequency. To measure recombination between these genes, we must set up a merozygote that is heterozygous for all three. This can be accomplished if we can establish which gene enters last by an interruptedconjugation analysis. The Hfr allele of the last-entering gene is selected among the F exconjugants. Then, knowing that we have selected the last gene, we know that the other two must also have been in the merozygote. If we know from interrupted-conjugation experiments that the gene order is met first followed by arg and then leu, the merozygote in a cross of Hfr met+ arg+ leu+ × F met arg leu must have been as follows:

The last gene to enter is leu+; therefore we select initially for leu+ exconjugants by plating them on medium containing no leucine but containing methionine and arginine. Now we can proceed to calculate map distance in the standard way by using a map unit equal to a recombinant frequency of 1 percent. In practice, this calculation is done by measuring the proportion of the total leu+ exconjugants that also carries arg+ or met+ or both or neither. The recombination events needed to produce these recombinant genotypes are shown in Figure 9-10. We know that a double crossover must have occurred to integrate leu+: one crossover is at the left of the leu gene, but the other can be in various positions at the right. Hence the genotype that arises from recombination between leu and arg will be leu+ arg met; so the percentage of bacteria with this genotype in the leu+ exconjugants will give us our recombinant frequency value for the leu-to-arg interval. The leu+ exconjugants arising from recombination between met and arg will be leu+ arg+ met. The percentage of bacteria with this leu+ subgenotype will provide the recombinant frequencies and hence the map distances between the genes.

In the cross just described, the leu+ arg met+ recombinants would require four crossovers instead of two (Figure 9-10d). These recombinants would be relatively rare.

Let us consider some data from this cross. The percentages of the three main genotypes obtained after testing leu+ exconjugants are:

From these results, we can conclude that the leuarg distance is 4 map units and that the argmet distance is 9 map units.

MESSAGE
Time-of-entry measurements in interrupted conjugation can generate a broad-scale map of the bacterial chromosome. Recombinant frequencies among exconjugants can be used in fine-scale mapping.

F Factors Carrying Bacterial Genes

Occasionally, the integrated F factor of an Hfr strain exits from the bacterial chromosome. Usually this event is a clean excision regenerating an intact F plasmid. However, as illustrated in Figure 9-5a, in some cases, the excision event is not a precise reversal of the original insertion, and a part of the bacterial chromosome is incorporated into the liberated plasmid. Figure 9-11 shows incorporation of a nearby lac gene into the plasmid, but the precise gene incorporated depends on where the F factor had originally integrated in the particular Hfr. Such plasmids carrying bacterial genes are called F. They are named for the gene that they carry: F-lac, as in the case illustrated in Figure 9-11, or F-gal, F-trp, and so forth. An F can be obtained by looking for rapid infectious transfer of a gene that is normally transferred late on the chromosome of the particular Hfr strain used.

If an F plasmid is transferred upon conjugation with an F strain, the recipients generated are stable merozygotes, carrying a complete bacterial genome plus a donor fragment on the autonomously replicating plasmid. The process of creating a merozygote by an F element is called sexduction or F-duction. Stable partial diploids are useful in bacterial genetics because they can be used for genetic studies usually possible only in a diploid cell, such as determination of dominance. For example, if a lac+ donor is used to create an F-lac+ plasmid and this plasmid is transferred to an F recipient that carries the allele lac, then the partial diploid is heterozygous lac+ / lac, and these cells can be used to determine which allele is dominant (lac+ turns out to be dominant in this case).

© 1999 by W. H. Freeman and Company. All rights reserved.