This section and subsequent sections describe the discovery of gene transfer in bacteria and explain several types of gene transfer and their use in bacterial genetics. First, we shall consider conjugation, which requires cell-to-cell contact. Conjugation was the first extensively studied method of gene transfer.
Discovery of conjugation
Do bacteria possess any processes similar to sexual reproduction and recombination? The question was answered in 1946 by the elegantly simple experimental work of Joshua Lederberg and Edward Tatum, who studied two strains of Escherichia coli with different nutritional requirements. Strain A would grow on a minimal medium only if the medium were supplemented with methionine and biotin; strain B would grow on a minimal medium only if it were supplemented with threonine, leucine, and thiamine. Thus, we can designate strain A as met−bio−thr+leu+thi+ and strain B as met+bio+thr−leu−thi−. Figure 7-2a displays in simplified form the concept of their experiment. Here, strains A and B are mixed together, and some of the progeny are now wild type, having regained the ability to grow without added nutrients. Figure 7-2b illustrates their experiment in more detail.
Demonstration by Lederberg and Tatum of genetic recombination between bacterial cells. Cells of type A or type B cannot grow on an unsupplemented (minimal) medium (MM), because A and B each carry mutations that cause the inability to synthesize constituents (more...)
Lederberg and Tatum plated bacteria into dishes containing only unsupplemented minimal medium. Some of the dishes were plated only with strainA bacteria, some only with strain B bacteria, and some with a mixture of strain A and strain B bacteria that had been incubated together for several hours in a liquid medium containing all the supplements. No colonies arose on plates containing either strain A or strain B alone, showing that back mutations cannot restore prototrophy, the ability to grow on unsupplemented minimal medium. However, the plates that received the mixture of the two strains produced growing colonies at a frequency of 1 in every 10,000,000 cells plated (in scientific notation, 1 × 10−7). This observation suggested that some form of recombination of genes had taken place between the genomes of the two strains to produce prototrophs.
Requirement for physical contact
It could be suggested that the cells of the two strains do not really exchange genes but instead leak substances that the other cells can absorb and use for growing. This possibility of “cross feeding” was ruled out by Bernard Davis. He constructed a U-tube in which the two arms were separated by a fine filter. The pores of the filter were too small to allow bacteria to pass through but large enough to allow easy passage of the fluid medium and any dissolved substances (Figure 7-3). Strain A was put in one arm; strain B in the other. After the strains had been incubated for a while, Davis tested the content of each arm to see if cells had become able to grow on a minimal medium, and none were found. In other words, physical contact between the two strains was needed for wild-type cells to form. It looked as though some kind of gene transfer had taken place, and genetic recombinants were indeed produced.
Experiment demonstrating that physical contact between bacterial cells is needed for genetic recombination to take place. A suspension of a bacterial strain unable to synthesize certain nutrients is placed in one arm of a U-tube. A strain genetically (more...)
Discovery of the fertility factor (F)
In 1953, William Hayes determined that genetic transfer occurred in one direction in the above types of crosses. Therefore, the transfer of genetic material in E. coli is not reciprocal. One cell acts as donor, and the other cell acts as the recipient. This kind of unidirectional transfer of genes was originally compared to a sexual difference, with the donor being termed “male” and the recipient “female.” However, this type of gene transfer is not true sexual reproduction. In bacterial gene transfer, one organism receives genetic information from a donor; the recipient is changed by that information. In sexual reproduction, two organisms donate equally (or nearly so) to the formation of a new organism, but only in exceptional cases is either of the donors changed.
The transfer of genetic material in E. coli is not reciprocal. One cell acts as the donor, and the other cell acts as the recipient.
Loss and regain of ability to transfer.
By accident, Hayes discovered a variant of his original donor strain that would not produce recombinants on crossing with the recipient strain. Apparently, the donor-type strains had lost the ability to transfer genetic material and had changed into recipient-type strains. In his analysis of this “sterile” donor variant, Hayes realized that the fertility (ability to donate) of E. coli could be lost and regained rather easily. Hayes suggested that donor ability is itself a hereditary state imposed by a fertility factor (F). Strains that carry F can donate, and are designated F+. Strains that lack F cannot donate and are recipients. These strains are designated F−.
Transfer of F during conjugation
Recombinant genotypes for marker genes are relatively rare in bacterial crosses, Hayes noted, but the F factor apparently was transmitted effectively during physical contact, or conjugation. A kind of “infectious transfer” of the F factor seemed to be taking place. We now know much more about the process of conjugation and about F, which is an example of a plasmid that can replicate in the cytoplasm independently of the host chromosome. Figures 7-4 and 7-5 show how bacteria can transfer plasmids such as F. The F plasmid directs the synthesis of pili, projections that initiate contact with a recipient (Figure 7-4) and draw it closer, allowing the F DNA to pass through a pore into the recipient cell. One strand of the double-stranded F DNA is transferred and then DNA replication restores the complementary strand in both the donor and the recipient. This replication results in a copy of F remaining in the donor and another appearing in the recipient, as shown in Figure 7-5.
Bacteria can transfer plasmids (circles of DNA), through conjugation. A donor cell extends one or more projections—pili—that attach to a recipient cell and pull the two bacteria together. (Oliver Meckes/MPI-Tübingen, Photo Researchers.) (more...)
(a) During conjugation, the pilus pulls two bacteria together. (b) Next, a bridge (essentially a pore) forms between the two cells. Then one strand of plasmid DNA passes into the recipient bacterium, and each single strand becomes double stranded again. (more...)
An important breakthrough came when Luca Cavalli-Sforza discovered a derivative of an F+strain. On crossing with F− strains this new strain produced 1000 times as many recombinants for genetic markers as did a normal F+ strain. Cavalli-Sforza designated this derivative an Hfr strain to indicate a high frequency of recombination. In Hfr × F− crosses, virtually none of the F− parents were converted into F+ or into Hfr. This result is in contrast with F+ × F− crosses, where infectious transfer of F results in a large proportion of the F− parents being converted into F+. Figure 7-6 portrays this concept. It became apparent that an Hfr strain results from the integration of the F factor into the chromosome, as pictured in Figure 7-6a.
The transfer of E. coli chromosomal markers mediated by F. (a) Occasionally, the independent F factor combines with the E. coli chromosome. (b) When the integrated F transfers to another E. coli cell during conjugation, it carries along any E. coli DNA (more...)
Now, during conjugation between an Hfr cell and a F− cell a part of the chromosome is transferred with F. Random breakage interrupts the transfer before the entire chromosome is transferred. The chromosomal fragment can then recombine with the recipient chromosome. Clearly, the low level of chromosomal marker transfer observed by Lederberg and Tatum (see Figure 7-2) in an F+ × F−cross can be explained by the presence of rare Hfr cells in the population. When these cells are isolated and purified, as first done by Cavalli, they now transfer chromosomal markers at a high frequency, because every cell is an Hfr.
Determining linkage from interrupted-mating experiments
The exact nature of Hfr strains became clearer in 1957, when Elie Wollman and François Jacob investigated the pattern of transmission of Hfr genes to F− cells during a cross. They crossed Hfr strs a+ b+ c+ d+ with F−strr a− b− c− d−. At specific time intervals after mixing, they removed samples. Each sample was put in a kitchen blender for a few seconds to disrupt the mating cell pairs and then was plated onto a medium containing streptomycin to kill the Hfr donor cells. This procedure is called interrupted mating. The strr cells then were tested for the presence of marker alleles from the donor. Those strr cells bearing donor marker alleles must have taken part in conjugation; such cells are called exconjugants.Figure 7-7a shows a plot of the results; azir, tonr, lac+, and gal+ correspond to the a+, b+, c+, and d+ mentioned in our generalized description of the experiment. Figure 7-7b portrays the transfer of markers.
Interrupted-mating conjugation experiments with E. coli. F− cells that are strr are crossed with Hfr cells that are strs. The F− cells have a number of mutations (indicated by the genetic markers azi, ton, lac, and gal) that prevent them (more...)
The most striking thing about these results is that each donor allele first appeared in the F− recipients at a specific time after mating began. Furthermore, the donor alleles appeared in a specific sequence. Finally, the maximal yield of cells containing a specific donor allele was smaller for the donor markers that entered later. Putting all these observations together, Wollman and Jacob concluded that gene transfer occurs from a fixed point on the donor chromosome, termed the origin (O), and continues in a linear fashion.
The Hfr chromosome, originally circular, unwinds and is transferred to the F− cell in a linear fashion. The unwinding and transfer begin from a specific point at one end of the integrated F, called the origin or O. The farther a gene is from O, the later it is transferred to the F−; the transfer process most likely will stop before the farthermost genes are transferred.
Wollman and Jacob realized that it would be easy to construct linkage maps from the interrupted-mating results, using as a measure of “distance” the times at which the donor alleles first appear after mating. The units of distance in this case are minutes. Thus, if b+ begins to enter the F− cell 10 minutes after a+ begins to enter, then a+ and b+ are 10 units apart (Figure 7-8). Like the maps based on crossover frequencies, these linkage maps are purely genetic constructions; at the time, they had no known physical basis.
Chromosome map of Figure 7-7. A linkage map can be constructed for the E. coli chromosome from interrupted-mating studies by using the time at which the donor alleles first appear after mating. The units of distance are given in minutes; arrowhead at (more...)
Chromosome circularity and integration of F
When Wollman and Jacob allowed Hfr × F− crosses to continue for as long as 2 hours before blending, they found that a few of the exconjugants were converted into Hfr. In other words, an important part of F (the terminal part now known to confer “maleness,” or donor ability), was eventually being transmitted but at a very low efficiency, and it apparently was transmitted as the last element of the linear chromosome. We now have the following map, in which the arrow indicates the process of transfer, beginning with O:
However, when several different Hfr linkage maps were derived by interrupted-mating and time-of-entry studies using different, separately derived Hfr strains, the maps differed from strain to strain.
At first glance, there seems to be a random reshuffling of genes. However, a pattern does exist; the genes are not thrown together at random in each strain. For example, note that in every case the hisgene has gal on one side and gly on the other. Similar statements can be made about each gene, except when it appears at one end or the other of the linkage map. The order in which the genes are transferred is not constant. In two Hfr strains, for example, the his gene is transferred before the gly gene (his is closer to O), but, in three strains, the gly gene is transferred before the his gene.
How can we account for these unusual results? Allan Campbell proposed a startling hypothesis: suppose that, in an F+ male, F is a small cytoplasmic element (and therefore easily transferred to an F− cell on conjugation). If the chromosome of the F+ male were a ring, any of the linear Hfr chromosomes could be generated simply by inserting F into the ring at the appropriate place and in the appropriate orientation (Figure 7-9).
Circularity of the E. coli chromosome. (a) Through the use of different Hfr strains (H, 1, 2, 3, 312) that have the fertility factor inserted into the chromosome at different points and in different directions, interrupted-mating experiments indicate (more...)
Several conclusions—later confirmed—follow from this hypothesis.
The orientation in which F is inserted would determine the polarity of the Hfr chromosome, as indicated in Figure 7-9a.
At one end of the integrated F factor would be the origin, where transfer of the Hfr chromosome begins; the terminus at the other end of F would not be transferred unless all the chromosome had been transferred. Because the chromosome often breaks before all of it is transferred and because the F terminus is what confers maleness, then only a small fraction of the recipient cells would be converted into male cells.
How, then, might F integration be explained? Wollman and Jacob suggested that some kind of crossover event between F and the F+chromosome might generate the Hfr chromosome. Campbell then came up with a brilliant extension of that idea. He proposed that, if F, like the chromosome, were circular, then a crossover between the two rings would produce a single larger ring with F inserted (Figure 7-10).
Now suppose that F consists of three different regions, as shown in Figure 7-10. If the bacterial chromosome had several homologous regions that could match up with the pairing region of F, then different Hfr chromosomes could be easily generated by crossovers at these different sites.
Chromosomal and F circularity were wildly implausible concepts initially, inferred solely from the genetic data; confirmation of their physical reality came only a number of years later. The direct-crossover model of integration also was subsequently confirmed.
The fertility factor thus exists in two states: (1) the plasmid state, as a free cytoplasmic element F that is easily transferred to F− recipients, and (2) the integrated state, as a contiguous part of a circular chromosome that is transmitted only very late in conjugation. The word episome (literally, “additional body”) was coined for a genetic particle having such a pair of states. A cell containing F in the first state is called an F+ cell, a cell containing F in the second state is an Hfr cell, and a cell lacking F is an F− cell. Today the term plasmid is used to refer to any self-replicating circular element in the cytoplasm and “episome” is rarely used.
Insertion of the F factor into the E. coli chromosome by crossing-over. Hypothetical markers 1 and 2 are shown on F to depict the direction of insertion. The origin (O) is the mobilization point where insertion begins; the pairing region is homologous (more...)
A frightening ability of pathogenic bacteria was discovered in Japanese hospitals in the 1950s. Bacterial dysentery is caused by bacteria of the genus Shigella. This bacterium initially proved sensitive to a wide array of antibiotics that were used to control the disease. In the Japanese hospitals, however, Shigella isolated from patients with dysentery proved to be simultaneously resistant to many of these drugs, including penicillin, tetracycline, sulfanilamide, streptomycin, and chloramphenicol. This multiple-drug-resistance phenotype was inherited as a single genetic package, and it could be transmitted in an infectious manner—not only to other sensitive Shigella strains, but also to other related species of bacteria. This talent is an extraordinarily useful one for the pathogenic bacterium, and its implications for medical science were terrifying. From the point of view of the geneticist, however, the situation is very interesting. The vector carrying these resistances from one cell to another proved to be a self-replicating element similar to the F factor. These R factors (for “resistance”) are transferred rapidly on cell conjugation, much like the F particle in E. coli.
In fact, these R factors proved to be just the first of many similar F-like factors to be discovered. These elements, which exist in the plasmid state in the cytoplasm, have been found to carry many different kinds of genes in bacteria. Table 7-2 shows some of the characteristics that can be borne by plasmids.
Genetic Determinants Borne by Plasmids.
Mechanics of transfer
Does an Hfr cell die after donating its chromosome to an F− cell? The answer is no (unless the culture is treated with streptomycin). The Hfr chromosome replicates while it is transferring a single strand to the F− cell; this replication ensures a complete chromosome for the donor cell after mating. The transferred strand is replicated in the recipient cell, and donor genes may become incorporated in the recipient’s chromosome through crossovers, creating a recombinant cell. Otherwise, transferred fragments of DNA in the recipient are lost in the course of cell division.
We assume that the F−chromosome is also circular, because the recipient F− cell, if it receives the F factor from an F+ cell, is readily converted into an F+ cell from which an Hfr cell can be derived.
The picture emerges of a circular Hfr chromosome unwinding a copy of itself, which is then transferred in a linear fashion into the F− cell. How is the transfer achieved? Electron microscope studies show that Hfr and F+ cells have fibrous structures, F pili, protruding from their cell walls, as shown in Figure 7-4. The F pili facilitate cell-to-cell contact, during which DNA is transferred through pores in the F−.
E. coli conjugation cycle
We can now summarize the various aspects of the conjugation cycle in E. coli (Figure 7-11). We shall review the conjugation cycle in regard to the differences between F−, F+, and Hfr cells, because these differences epitomize the cycle.
F−strains do not contain the F factor and cannot transfer DNA by conjugation. They are, however, recipients of DNA transferred from F+ or Hfr cells by conjugation.
F+cells contain the F factor in the cytoplasm and can therefore transfer F in a highly efficient manner to F− cells during conjugation.
Hfr cells have F integrated into the bacterial chromosome, not in the cytoplasm.
Summary of the various events that take place in the conjugational cycle of E. coli.
Chromosomal markers are transferred in a strain of F+ cells because, in any population of F+ cells, a small fraction of cells (about 1 in 1000) have been converted into Hfr cells by the integration of F into the bacterial chromosome. Because conjugation experiments are usually carried out by mixing from 107 to 108 cells consisting of prospective donors and recipients, the population will contain various different Hfr cells derived from independent integrations of F into the chromosome at various different sites. Therefore, when chromosomal markers are transferred by different cells in the population, transfer will start at different points on the chromosome. This results in an approximately equal transfer of markers all around the chromosome, although at a low frequency. This type of F+-mediated transfer is what Lederberg and Tatum observed when they discovered gene transfer in bacteria.
Each of the Hfr cells in an F+ population with an integrated F factor can be the source of a new Hfr strain if it is isolated and used to start a clone.
Hfr strains are derived from a clone of Hfr cells in which a specific integration of F into the bacterial chromosome has taken place. Therefore, all the cells in any given Hfr strain have F integrated into the chromosome at exactly the same point.
Hfr populations transfer chromosomal markers to F− cells at a high frequency compared with F+ populations, because only a fraction of cells in an F+ population have F integrated into the chromosome. Further, in any given Hfr strain, the markers are transferred from a fixed point in a specific order. This also contrasts with F+ populations, where the Hfr cells transfer chromosomal markers in no particular fixed order, given that the F factor integrates into the chromosome at different points in different F+ cells.
In an Hfr × F−cross, the F− is not converted into Hfr or into F+, except in very rare cases, because the Hfr chromosome nearly always breaks before the F terminus is transferred to the F− cell.
Recombination between marker genes after transfer
Thus far, we have studied only the process of the transfer of genetic information between individuals in a cross. This transfer is inferred from the existence of recombinants produced from the cross. However, before a stable recombinant can be produced, the transferred genes must be integrated or incorporated into the recipient’s genome by an exchange mechanism. We now consider some of the special properties of this exchange event.
Genetic exchange in prokaryotes does not take place between two whole genomes (as it does in eukaryotes); rather, it takes place between one completegenome, derived from F−, called the endogenote, and an incomplete one, derived from the donor, called the exogenote. What we have in fact is a partial diploid, or merozygote. Bacterial genetics is merozygous genetics. Figure 7-12a is a diagram of a merozygote.
Crossover between exogenote and endogenote in a merozygote. (a) The merozygote. (b) A single crossover leads to a partly diploid linear chromosome. (c) An even number of crossovers leads to a ring plus a linear fragment.
A single crossover would not be very useful in generating viable recombinants, because the ring is broken to produce a strange, partly diploid linear chromosome (Figure 7-12b). To keep the ring intact, there must be an even number of crossovers (Figure 7-12c). The fragment produced in such a crossover is only a partial genome, which is generally lost in subsequent cell growth. Hence, both reciprocal products of recombination do not survive—only one does. A further unique property of bacterial exchange, then, is that we must forget about reciprocal exchange products in most cases.
In the genetics of bacteria, we generally are concerned with double crossovers and we do not expect reciprocal recombinants.
Gradient of transfer
Only partial diploids exist in the merozygote. Some genes don’t even get into the act. To better understand this fact, let us look again at the consequences of gene transfer. Usually, only a fragment of the donor chromosome appears in the recipient, owing to spontaneous breakage of the mating pairs; so the entire chromosome is rarely transferred. The spontaneous breakage can occur at any time after transfer begins, which creates a natural gradient of transfer and makes it less and less likely that a recipient cell will receive later and later genetic markers. (“Later” here refers to markers that are increasingly farther from the origin and hence are donated later in the order of markers transferred.) For example, in a cross of Hfr-donating markers in the order met, arg, leu, we would expect a distribution of fragments such as the one represented here:
Note that many more fragments contain the met locus than the arg locus and that the leu locus is present on only one fragment. It is easy to see that the closer the marker is to the origin, the greater the chance it will be transferred in conjugation.
The concept of the gradient of transfer is the same as the one described earlier for interrupted matings, except that here we are allowing the natural disruption of mating pairs to occur instead of interrupting the pairs mechanically.
Determining gene order from gradient of transfer
We can use the natural gradient of transfer to establish the order of genetic markers, provided we select for an early marker that enters before the markers that we are ordering. Let’s see how this works. Suppose that we use an Hfr strain that donates markers in the order met, arg, aro, his. In a cross of an Hfr that is met+ arg+ aro+ his+ strs with an F− that is met− arg− aro− his− strr, recombinants are selected that can grow on a minimal medium without methionine but with arginine, aromatic amino acids, and histidine and in the presence of streptomycin. Here we are selecting for recombinants in the F− strain that are met+ in a cross in which the met locus is transferred as the earliest marker. We can then score for inheritance of the other markers present in the Hfr by testing on supplemental minimal medium lacking, in turn, one of the required nutrients.
A typical result would be:
Note how the frequency of inheritance corresponds to the order of transfer. This correspondence is due to the fact that the frequency of inheritance is indicative of the frequency of transfer. For this method to work, it is crucial that it be applied only to genetic markers that enter after the selected marker—in this case, after met.
Higher-resolution mapping by recombinant frequency in bacterial crosses
Although interrupted-mating experiments and the natural gradient of transfer can give us a rough set of gene locations over the entire map, other methods are needed to obtain a higher resolution between marker loci that are close together. Here we consider one approach to the problem: using the frequency of recombinants to measure linkage.
Previous attempts to measure linkage in conjugational crosses were hindered by the failure to understand that only fragments of the chromosome are transferred and that the gradient of transfer produces a bias toward the inheritance of early markers. To measure linkage and to attach any meaning to a calculated map distance, it is necessary to produce a situation in which every marker has an equal chance at being transferred so that the recombinant frequencies are dependent only on the distance between the relevant genes.
Suppose that we consider three markers: met, arg, and leu. If the order is met, arg, leu and if met is transferred first and leu last, then we want to set up the situation diagrammed here to calculate map distances separating these markers:
Here, we have to arrange to select the lastmarker to enter, which in this case is leu. Why? Because, if we select for the last marker, then we know that every cell that received fragments containing the last marker also received the earlier markers—namely, arg and met—on the same fragments. We can then proceed to calculate map distance in the classic manner. Rather than using map units, we simply refer to the percentage of crossovers in the respective interval on the map. In practice, this is done by calculating, among the total recovered recombinants, the percentage of recombinants produced by crossovers between two markers. Let’s look at an example.
In the cross of the Hfr strain just described (met+ arg+ leu+ strs) with an F− that is met− arg− leu− strr, we would select leu+ recombinants and then examine them for the arg and met markers. In this case, the arg and met markers are called the unselected markers.Figure 7-13 depicts the types of crossover events expected. Note how two crossover events are required to incorporate part of the incoming fragment into the F−chromosome. One crossover must be on each side of the selected (leu)marker. Thus, in Figure 7-13, one crossover must be on the left side of the leu marker and the second must be on the right side. Suppose that the map distance between each marker is 5 percent recombination. In 5 percent of the total leu+ recombinants, the second crossover occurs between leu and arg (Figure 7-13a); in another 5 percent of the cases, the second crossover occurs between leu and met (Figure 7-13b). We would then expect 90 percent of the selected leu+ recombinants to be arg+ met+, because the second crossover occurs outside the leu–arg–met interval (Figure 7-13c) in 90 percent of the cases. We would also expect 5 percent of the leu+ recombinants to be arg− met−, resulting from a crossover between leu and arg, and 5 percent of the leu+ recombinants to be arg+ met−, resulting from a crossover between arg and met. In reality, then, we are simply determining the percentage of the time that the second crossover occurs in each of the three possible intervals.
Incorporation of a late marker into the F−E. coli chromosome. After an Hfr cross, selection is made for the leu+ marker, which is donated late. The early markers (arg+ and met+) may or may not be inserted, depending on the site where recombination (more...)
In a cross such as the one just described, one class of potential recombinants requires an additional two crossover events (Figure 7-13d). In this case, the leu+ arg− met+ recombinants would require four crossovers instead of two. These recombinants are rarely recovered, because their frequency is sharply reduced compared with the other classes of recombinants.
Infectious marker-gene transfer by episomes
Edward Adelberg’s work led to the discovery of gene transfer at high frequency by episomes. When he began his recombination experiments in 1959, the particular Hfr strain that he used kept producing F+ cells, so the recombination frequencies were not very large. Adelberg called this particular fertility factor F′ to distinguish it from the normal F, for the following reasons:
The F′-bearing F+strain reverted back to an Hfr strain much more frequently than do typical F+ strains.
F′ always integrated at the same place to give back the original Hfr chromosome. (Remember that randomly selected Hfr derivatives from F+ males have origins at many different positions.)
How could these properties of F′ be explained? The answer came from the recovery of an F′ from an Hfr strain in which the lac+ locus was near the end of the Hfr chromosome (it was transferred very late). Using this Hfr lac+ strain, François Jacob and Adelberg found an F+ derivative that transferred lac+ to F−lac− recipients at a very high frequency. Furthermore, the recipients that behaved like F+lac+ occasionally produced F−lac−daughter cells, at a frequency of 1 × 10−3. Thus, the genotype of these recipients appeared to be F lac+/lac−.
Now we have the clue: F′ is a cytoplasmic element that carries a part of the bacterial chromosome. In fact, it is nothing more than F with a piece of the host chromosome incorporated. Its origin and reintegration can be visualized as shown in Figure 7-14. This F′ is known as F′ lac, because the piece of host chromosome that it picked up has the lacgene on it. F′ factors have been found carrying many different chromosomal genes and have been named accordingly. For example, F′ factors carrying gal or trp are called F′ gal and F′ trp, respectively. Because F lac+/lac− cells are Lac+ in phenotype, we know that lac+ is dominant over lac−. As we shall see in Chapter 11, the dominant– recessive relation between alleles can be a very useful bit of information in interpreting gene function.
Origin and reintegration of the F′ factor, in this case, F′ lac. (a) F is inserted in an Hfr strain between the ton and lac+ alleles. (b and c) Abnormal “outlooping” and separation of F occurs to include the lac locus, (more...)
Partial diploidy for specific segments of the genome can be made with an array of F′ derivatives from Hfr strains. The F′ cells can be selected by looking for the infectious transfer of normally late genes in a specific Hfr strain. Some F′ strains can carry very large parts (up to one-quarter) of the bacterial chromosome; if appropriate markers are used, the merozygotes generated can be used for recombination studies.
During conjugation between an Hfr donor and an F− recipient, the genes of the donor are transmitted linearly to the F− cell, through the bacterial chromosome, with the inserted fertility factor transferring last.
In the course of conjugation between an F+ donor carrying an F′ plasmid and an F− recipient, a specific part of the donor genome may be transmitted infectiously to the F− cell, through the plasmid. The transmitted part was originally adjacent to the F locus in an Hfr strain from which the F+ was derived.
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 DNAplasmid 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:
The F plasmid can replicate its own DNA, allowing the plasmid to be maintained in a dividing cell population (Figure 9-3a).
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).
F+ and F− cells can conjugate. When conjugation occurs, the F+ cells can act as F donors. The F plasmidDNA 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.
F+ cells are usually inhibited from making contact with other F+ cells; therefore the F plasmid is not transferred from F+ to F+.
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).
Some properties of the fertility (F) factor of E. coli.
The transfer of E. coli chromosomal markers mediated by F. (a) Occasionally, the independent F factor combines with the E. coli chromosome. (b) When the integrated F transfers to another E. coli cell during conjugation, it carries along any E. coli DNA (more...)
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).
Genetics In Process 9-1: Lederberg and Tatum discover genetic recombination in bacteria. In eukaryotes, the sexual cycle brings together genomes from two parents into one zygotic cell; then, in this cell or in descendent cells, meiosis takes place, which (more...)
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.
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 gene—one 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).
Transfer of single-stranded fragment of donor chromosome, and recombination with recipient chromosome. note: double crossovers can occur in any location; those shown are examples.
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 allelea+ 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 azirgene 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.
Interrupted-mating conjugation experiments with E. coli. F− cells that are strr are crossed with Hfr cells that are strs. The F− cells have a number of mutations (indicated by the genetic markers azi, ton, lac, and gal) that prevent then (more...)
Chromosome map based on Figure 9-7. A linkage map can be constructed for the E. coli chromosome from interrupted-mating studies, by using the time at which the donor alleles first appear after mating. The units of distance are given in minutes; arrowhead (more...)
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.
Circularity of the E. coli chromosome. (a) Through the use of different Hfr strains (H, 1, 2, 3, 312) that have the fertility factor inserted into the chromosome at different points and in different directions, interrupted-mating experiments indicate that (more...)
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 genes—met, arg, and leu—by 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.
Mapping by recombination in E. coli. After a cross, selection is made for the leu+ marker, which is donated late. The early markers (arg+ and met+) may or may not be inserted, depending on the site where recombination between the Hfr fragment and the (more...)
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 leu–arg distance is 4 map units and that the arg–met distance is 9 map units.
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 lacgene 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.
Origin and reintegration of the F′ factor—in this case, F′ lac. (a) F is inserted in an Hfr strain between the ton and lac+ alleles. (b,c) Abnormal “outlooping” and separation of F occurs to include the lac locus, (more...)
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 allelelac−, 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).