FIGURE 1.

Specialized transduction is outlined as follows: the center plasmid represents the shuttle phasmid phA159, which contains 90% TM4 phage DNA and 10% plasmid DNA. The stars mark the sites of the mutations in the TM4 genome. The nonessential genes that are deleted to create the shuttle phasmid are noted in the picture, flanked by PacI sites. This site can be replaced with one of three things: (i) a reporter gene such as green fluorescent protein (GFP), (ii) an allelic exchange substrate (AES) that contains an antibiotic resistance marker, or (iii) a transposase gene to facilitate transposon mutagenesis. Going counterclockwise in this schematic, the recombinant cosmid can be packaged into phage heads using an in vitro packaging mix, and the subsequent phages can be used to transduce E. coli to create E. coli transductant colonies. Going clockwise from the shuttle phasmid, one can transfect M. smegmatis mc²155 at 30°C to yield plaques on an M. smegmatis lawn, resulting from lysis of the cells. The plaques can then be purified and amplified to obtain a high-titer phage lysate that can subsequently be used to transduce any mycobacterial species. doi:10.1128/microbiolspec.MGM2-0037-2013.f1