Fig. 3

Gene deletion and integration via homologous recombination into the genome of E. mundtii ST4SA at the munA bacteriocin gene locus to create E. mundtii ST4SA munA::cat-ffluc. a Homologous recombination between the wild-type (WT) E. mundtii ST4SA munA-carrying megaplasmid and the munA::catffluc cassette. Boxed regions show the upstream (~ 0.9 kb) and downstream regions (~ 0.6) of homology on the megaplasmid and the pNZKOmunACatFfluc knockout (KO) vector. Cells harboring the munA KO vector were selected on Cm and Em, followed by nisin induction for MazF toxin expression to select for mutants that have lost the plasmid backbone bearing erm and mazF genes. Double crossover mutants were selected and screened by PCR using the indicated primer combinations. b PCR amplification of WT and munA deletion and insertion mutants using the primer pair indicated in panel (a). Primer pairs are shown in purple. (m) Lambda DNA digested with PstI (NEB). Amplicons from four munA mutant and two WT colonies, respectively, are shown