Fig. 5.

Gene deletion and integration via homologous recombination into the genome of E. mundtii ST4SA at the srtA locus to create E. mundtii ST4SA srtA::FRTerm, and E. mundtii ST4SA sortase A aggregation substance (AS) cell clumping assay. a Schematic representing the wild-type (WT) E. mundtii ST4SA srtA gene locus and the recombinant srtA deletion and FRT-erm integration site. Boxed regions show the upstream and downstream regions of homology (~ 1 kb) on the WT chromosome and the recombinant srtA::FRTerm locus. Cells harboring the srtA knockout 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 cat and mazF genes. Double crossover mutants were selected and screened by PCR using the primer combinations shown in purple. b PCR amplification of WT and srtA deletion and insertion mutants using the primer pair indicated in panel A. (m) Lambda DNA digested with PstI (NEB). Amplicons from one WT and two srtA::FRTerm insertion mutant colonies are shown. c MRS broth with the WT strain containing SrtA AS. d MRS broth containing the E. mundtii ST4SA srtA::FRTerm deletion mutant strain lacking SrtA AS. e MRS broth with E. mundtii ST4SA srtC::FRTerm deletion mutant strain containing SrtA AS