Fig. 1.

Schematic representing the pNZmazFnisRK inducible mazF toxin vector used for the construction of double-crossover homologously recombined DNA integration or deletion mutants at any genomic loci. Relevant features are indicated, including restriction sites used for cloning; the E. coli/LAB repA and repC replication genes; the chloramphenicol acetyltransferase (cat) gene conferring resistance to chloramphenicol; the nisR and nisK nisin regulatory genes; and the nisin-inducible PnisA promoter from Lc. lactis pNZ9000. Integration cassettes are inserted via blunting into the BglII, HindIII or StuI restriction sites