FIG 6.

pMBsacB permits efficient generation of unmarked mutations in the cylE gene. (A) After generation of a cylE mutagenesis cassette bearing a premature stop codon barcoded by additional silent SNPs, the cassette was cloned into pMBsacB and used to knockout β-hemolysin/cytolysin expression in GBS 10/84 and A909. (B) Sucrose counterselection resulted in near-complete elimination of erythromycin-resistant CFU. (C) Clones that survived counterselection were screened for the barcoded mutation by performing PCR with cylE_farout_F/R primers and Sanger sequencing of the amplified genomic region of interest with cylEmut_Conf_F. Representative traces are shown for the bands marked by asterisks. (D) The recovered knockout strains had the expected nonhemolytic phenotype when plated on 5% sheep’s blood agar. Erm, erythromycin.