Transduction Using Phages to Map Bacterial Genes

In the discussion of bacterial genetics, we identified three mechanisms of gene transfer: conjugation, transformation, and transduction (see Figure 8.9). Let's take a closer look at transduction, in which genes are transferred between bacte-

Question: Does genetic exchange between bacteria always require cell-to-cell contact?

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^ Two auxotrophic strains of Salmonella typhimurium were mixed.

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^ Two auxotrophic strains of Salmonella typhimurium were mixed.

When the two strains were placed in a Davis U-tube,...

When the two strains were placed in a Davis U-tube,...

Filter i.

. and plated on minimal medium.

^ .which separated the strains by a filter with pores too small for the bacteria to pass through,.

Prototrophic colonies

No colonies

I Some prototrophic colonies were obtained.

I Some prototrophic colonies were obtained.

^ .prototrophic colonies were obtained from only one side of the tube.

Conclusion: Genetic exchange did not take place via conjugation. A phage was later shown to be the agent of transfer. n--

8.26 The Lederberg and Zinder experiment.

ria by viruses. In generalized transduction, any gene may be transferred. In specialized transduction, only a few genes are transferred.

Generalized transduction Joshua Lederberg and Norton Zinder discovered generalized transduction in 1952. They were trying to produce recombination in the bacterium Salmonella typhimurium by conjugation. They mixed a strain of S. typhimurium that was phe+ trp+ tyr+ met~ his~ with a strain that was phe~ trp~ tyr~ met+ his+ (< Figure 8.26) and plated them on minimal medium. A few prototrophic recombinants (phe+ trp+ tyr+ met+ his+) appeared, suggesting that conjugation had taken place. However, when they tested the two strains in a U-shaped tube similar to the one used by Davis, some phe+ trp+ tyr+ met+ his+ prototrophs were obtained on one side of the tube (compare Figure 8.26 with Figure 8.11). This apparatus separated the two strains by a filter with pores too small for the passage of bacteria; so how were genes being transferred between bacteria in the absence of conjugation? The results of subsequent studies revealed that the agent of transfer was a bacteriophage.

In the lytic cycle of phage reproduction, the bacterial chromosome is broken into random fragments ( FIGURE 8.27). For some types of bacteriophage, a piece of the bacterial chromosome occasionally gets packaged into a phage coat instead of phage DNA; these phage particles are called transducing phages. The transducing phage infects a new cell, releasing the bacterial DNA, and the introduced genes may then become integrated into the bacterial chromosome by a double crossover. Bacterial genes can, by this process, be moved from one bacterial strain to another, producing recombinant bacteria called transductants.

Not all phages are capable of transduction, a rare event that requires (1) that the phage degrade the bacterial chromosome; (2) that the process of packaging DNA into the phage protein not be specific for phage DNA; and (3) that the bacterial genes transferred by the virus recombine with the chromosome in the recipient cell.

Because of the limited size of a phage particle, only about 1% of the bacterial chromosome can be transduced. Only genes located close together on the bacterial chromosome will be transferred together (cotransduced). The overall rate of transduction ranges from only about 1 in 100,000 to 1 in 1,000,000. Because the chance of a cell being transduced by two separate phages is exceedingly small, any cotransduced genes are usually located close together on the bacterial chromosome. Thus, rates of cotransduction, like rates of cotransformation, give an indication of the physical distances between genes on a bacterial chromosome.

To map genes by using transduction, two bacterial strains with different alleles at several loci are used. The donor strain is infected with phages ( FIGURE 8.28), which reproduce within the cell. When the phages have lysed the donor cells, a suspension of the progeny phage is mixed with a recipient strain of bacteria, which are then plated on several different kinds of media to determine the pheno-

types of the transducing progeny phages. __

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