A total of 141 S. pyogenes strains belonging to 21 emm genotypes were analyzed. These included 138 strains obtained from patients with uncomplicated S. pyogenes infections, VEGFR inhibitor two strains isolated from patients with STSS, and one strain isolated from a sepsis patient. All strains were isolated between 1994 and 2006 in Toyama or Aichi Prefecture, Japan. emm genotypes were determined for all 141 strains according to the emm genotyping protocol (http://www.cdc.gov/ncidod/biotech/strep/strepindex.html). S. pyogenes SF370 (12) was included in all

examinations. A nonpolar inactivated mutant of the emm1 gene (SF370 Δemm1) was constructed in the chromosome of S. pyogenes SF370 through double-crossover allelic replacement. DNA fragments of emm1 were Metformin in vivo amplified with the oligonucleotide primers emm-n5Nhe and emm-c4Sma (fragment 1) and emm-n6Sma and emm-c5Spe (fragment 2). The primers used in this study are shown in Table 1. NheI/SmaI-digested fragment 1 was inserted in the same site in pFW12 (13). The resultant plasmid was digested with SmaI and SpeI, and both SmaI/SpeI-digested fragment

2 and an spc2 DNA fragment containing aad9 (promoterless spectinomycin resistance gene) obtained from an SmaI-digested fragment of pSL60-2 (13) were inserted. This plasmid (emm1::aad9/pFW12) was a suicide vector for S. pyogenes. For the preparation of competent cells, strain SF370 was harvested at early- to mid-log phase (OD660 = 0.4–0.5) and washed twice with 0.5 M sucrose buffer. The constructed suicide vector was transformed into the strain by electroporation, which was conducted at 1.25 kV/mm, 25 μF capacitance, and 200 ohms resistance using a Gene Pulser II (Bio-Rad Laboratories, Hercules, CA, USA). After incubation at 37°C for 3 hr, competent cells were spread onto BHIY on agar plates containing spectinomycin (final concentration, 100 μg/mL). Selected colonies on the plates were cultured. Cultured bacteria were washed once with saline, resuspended in 10 mM Tris–1 mM EDTA, and boiled for 10 Carnitine dehydrogenase min. Genomic DNA was obtained from

the supernatant of boiled bacteria. Double-crossover replacement with genomic DNA was analyzed by PCR. Successful double-crossover replacement was further confirmed by DNA sequencing. SF370 ΔcsrS was prepared according to a previously described method (14). The M protein-high producer of emm1 was complemented with the csrS (I333V) gene. One of our previous studies had demonstrated that, judging from the exoprotein production profile, the csrS (I333V) gene cannot be functionally distinguished from the wild type gene (15). Preparation of the csrS-complemented strain has been described previously (15). A homology search of four different emm genes (emm1, 3, 6, and 12) revealed that a fragment of 360 bps between the C2 and D repeat regions (amino acid position: 286–405 referenced to the SF370 genome strain) was identical in these genes.