FIG. 1.

Construction of fba mutants. The fba gene was amplified via PCR with genomic DNA isolated from GAS strain 90-226 as the template. The oligonucleotide primers were designed to create BamHI restriction sites at the ends of the amplified fragment. The amplicon was digested with BamHI, gel purified, and ligated to BamHI-digested pSportI to create the plasmid pSport-fba. A 730-bp HindIII-to-BglII fragment containing an internal fragment of fba was then subcloned into the suicide vector pFW5 (42). pFW5 carries the aad9 (spectinomycin resistance) gene and does not replicate in GAS. Plasmid pFW5Δfba was introduced into GAS strains 90-226 and 90-226 emm1::Km by electroporation. Southern blot and PCR analyses were performed to verify integration of pFW5Δfba into the chromosomal fba gene.