Mutant construction and integration vector-mediated gene complementation in Listeria monocytogenes

Genes that play role in stress response mechanisms and other phenotypes of bacteria can be identified by construction and screening of mutant libraries. In this chapter, we describe the construction and screening of mutant libraries of Listeria monocytogenes using a plasmid, pMC38, carrying a mariner-based transposon system (TC1/mariner) and constructed by Cao et al. (Appl Environ Microbiol 73:2758-2761, 2007). Following screening of the mutant library, putative mutants are identified and the transposon is localized, leading to identification of the genes that play possible roles in the phenotype of interest. To confirm the role of the gene in the relevant phenotype, transposon mutants are genetically complemented with the wild type gene using the site-specific temperature-sensitive integration vector pPL2, constructed by Lauer et al. (J Bacteriol 184:4177-4186, 2002).