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

Bacteriophages, the viruses that parasitize and kill bacteria, can be used in several different types of genetic analysis. First, two distinct phage genotypes can be crossed to measure recombination and hence map the viral genome. Second, they can be used as a way of bringing bacterial genes together for linkage and other genetic studies. These two uses will be described in this chapter. Finally, as we shall see in Chapter 10, phages are also used in molecular technology as carriers, or vectors, of foreign DNA inserts from any organism. First, we examine how a single phage attacks a bacterial cell.

Infection of Bacteria by Phages

Most bacteria are susceptible to attack by bacteriophages (the word literally means "eaters of bacteria"). A phage consists of a nucleic acid "chromosome" (generally DNA) surrounded by a coat of protein molecules. One well-studied class of phage strains comprises the T phages: T1, T2, and so forth. Figures 9-13 and 9-14 show the complicated structure of a phage belonging to the subclass called T-even (T2, T4, T6, and so forth).

During infection, a phage attaches to a bacterium and injects its genetic material into the bacterial cytoplasm (Figure 9-15a). The phage genetic information then takes over the machinery of the bacterial cell by turning off the synthesis of bacterial components and redirecting the bacterial synthetic system to make phage components (Figure 9-15b). (The use of the word information is interesting in this connection; it literally means "to give form." And that is precisely the role of the genetic material: to provide blueprints for the construction of form. In the present context, the form is the elegantly symmetrical structure of the new phages.) Ultimately, many phage descendants are released when the bacterial cell wall breaks open. This bursting process is called lysis.

But what phage phenotypes can we study routinely, given that they are so small that they are visible only under the electron microscope? In the study of phages, we cannot produce a visible colony by plating, but we can produce a visible manifestation of infected bacteria by taking advantage of several phage characters. Let's look at the consequences of a phage infecting a single bacterial cell. Figure 9-16 shows the sequence of events in the infectious cycle that leads to the release of progeny phages from the lysed cell. After lysis, the progeny phages infect neighboring bacteria, and these bacteria, too, lyse and reinfect. This reinfection results in an exponential increase in the number of lysed cells. Within 15 hours after the start of an experiment of this type, the effects are visible to the naked eye as a clear area, or plaque, on the opaque "lawn" of bacteria growing on the surface of a plate of solid medium (Figure 9-17). Such plaques can be large or small, fuzzy or sharp, and so forth, depending on the phage type. Thus, plaque morphology is a phage character that can be analyzed. Another phage character that we can analyze genetically is host range, because phages may differ in the spectrum of bacterial strains that they can infect and lyse. For example, certain strains of bacteria are immune to attachment or injection by specific phages.

The Phage Cross

Genetic analysis of any organism is accomplished by bringing together two genetically different genomes into the same cell and giving them an opportunity to engage in recombination and segregation. Likewise two distinct phage genomes can be united, but, because of the parasitic nature of phages, this union must be inside a bacterial cell. In other words, a phage cross must be made by a double infection of bacterial cells by the two phage types.

The procedure can be illustrated by a cross of T2 phages. The genotypes of the two parental strains are h r+ and h+ r. The alleles correspond to the following phenotypes: h can infect two different E. coli strains (which we can call strains 1 and 2); h+ can infect only one strain (strain 1); r rapidly lyses cells, thereby producing large plaques; and r+ slowly lyses cells, thus producing small plaques. In the cross, E. coli strain 1 is infected with both parental T2 phage genotypes at a phage:bacteria ratio that is high enough to ensure that a large percentage of cells are simultaneously infected by both phage types (Figure 9-18). The phage lysate (the progeny phages) is then analyzed by spreading it onto a bacterial lawn composed of a mixture of E. coli strains 1 and 2. Four plaque types are then distinguishable (Figure 9-19 and Table 9-2). These four genotypes can be scored easily as parental (h r+ and h+ r) and recombinant, and a recombinant frequency can be calculated as follows:

Analysis of phage crosses is subject to complications. First, several rounds of exchange can potentially occur within the host: a recombinant produced shortly after infection may undergo further exchange at later times. Second, double infection can occur between genetically similar phages as well as between different types. Thus, if we let P1 and P2 refer to parental phage genotypes, then crosses of the type P1 × P1 and P2 × P2 occur in addition to the informative cross P1 × P2. For both of these reasons, recombinants from phage crosses are a consequence of a population of events rather than defined, single-step exchange events. Nevertheless, all other things being equal, the RF calculation is a valid index of map distance in phages.

MESSAGE
Phage genomes can be mapped by analysis of recombinant frequency in double infections.

Lysogeny

Phages are categorized into two types. Virulent phages are always lytic; that is, they infect and lyse the host cell, resulting in progeny phages. In contrast, temperate phages can undergo a lytic cycle under some circumstances, but more often the phage integrates into the bacterial chromosome and henceforth the inserted phage is replicated along with the bacterial chromosome. In this condition, the phage is referred to as a prophage and the bacterial host is called lysogenic. A lysogenic bacterium, or lysogen, is resistant to subsequent infection, because an "immunity" is conferred by the presence of the prophage. The lysogenic state can be transmitted genetically through many bacterial generations. However, a prophage can occasionally excise from the bacterial chromosome and enter a lytic cycle, leading to lysis of its host cell and the production of a large number of progeny phages, referred to as a lysate. These progeny phages can infect and cause lysis in any nonlysogenic cells present in the same culture. (The word lysogenic means "lysis causing" as the property of self-immunity, but ability to lyse other genotypes was the way that this condition was first identified.)

MESSAGE
Virulent phages cannot become prophages; they are always lytic. Temperate phages can integrate into the bacterial chromosome as prophages, allowing the host cells to survive in the lysogenic state. Prophages occasionally exit the bacterial chromosome and enter the lytic cycle.

Further properties of lysogenic phage can be illustrated with the use of a temperate phage called lambda ( l ). Phage l , an E. coli phage, has become the most intensively studied and best-characterized phage. In a cross of a lysogenic Hfr with a nonlysogenic F recipient, the entry of the l prophage into the nonimmune cell immediately triggers the prophage into a lytic cycle; this event is called zygotic induction. But, in the cross Hfr( l ) × F( l ), conjugation proceeds normally, no lysis takes place, and bacterial recombinants are readily recovered. In other words, there is no zygotic induction followed by lysis (Figure 9-20). The cytoplasm of the F cell would seem to exist in two different states, depending on whether the cell contains a l prophage; contact between an entering prophage and the cytoplasm of a nonimmune cell would then immediately induce the lytic cycle. It is known that a cytoplasmic factor specified by the prophage represses the multiplication of the virus. Entry of the prophage into the environment of a nonlysogenic cell immediately dilutes this repressing factor, and therefore the virus reproduces. But, if the virus specifies the repressing factor, then why doesn't the virus shut itself off again? Clearly, it can, because a fraction of infected cells do become lysogenic. There is a race between the l gene signals for reproduction and those specifying a shutdown. The model of a phage-directed cytoplasmic repressor nicely explains the immunity of the lysogenic bacteria, because any superinfecting phage would immediately encounter a repressor and be inactivated.

The phenomenon of lysogeny is a way for a temperate phage to avoid eating itself out of house and home. Lysogenic cells can perpetuate and carry the phages relatively innocuously.

Phage Integration

How does the prophage integrate into the bacterial genome? The free form of the phage chromosome at this stage is circular. There is alignment of a specific site in l , the l attachment site, and a site in the bacterial chromosome located between the genes gal and bio. Then a reciprocal crossover takes place between the circular phage and the circular bacterial chromosome, resulting in the integration of the phage in the E. coli chromosome. The integration of the phage to become a prophage increases the genetic distance between flanking bacterial markers, as can be seen in Figure 9-21 for gal and bio. This integration is promoted by phage genes and not by the general bacterial recombination system.

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