Genetic Manipulations Using Phages

Lytic (or virulent) phages usually have only a single outcome upon infection of a bacterial host: reproduction of the virus and lysis of the bacterium. As a consequence, plaques appear as perfectly clear areas of infection, within which all of the bacterial cells are dead. The role of phage genetics in microbiology has expanded with the realization that we understand only a small fraction of bacterial diversity. Microbes such as Escherichia coli and Salmonella have been deeply studied and to great effect, but as our attention has turned to the broader bacterial community, new bacteria have brought new genetic challenges. This chapter describes some common phage methods and approaches that can be applied to virtually any bacterial host. Several collections of mycobacteriophages had been assembled previously with a to using their host-range specificities for the typing of clinical isolates. One of these phages had been shown to mediate generalized transduction in Mycobacterium smegmatis, and researchers have achieved uptake of phage DNA by M. tuberculosis. The chapter also describes how phages can be isolated, purified, and genomically characterized, how shuttle phasmids can be constructed and utilized, and how phages can be used to construct a variety of genetic tools. The potential for phage genetics in any bacterial host is vast, and the approaches and methods described in the chapter represent just a small part of what bacteriophages have to offer to the microbial genetic engineer.