TABLE 1.

Bacterial strains and plasmids

Strain or plasmidDescriptionReference
Strains
    GAS clinical isolates
        NZ131T14 OF+, emm49 glomerulonephritis isolate45
        5448T1 OF, emm1.0 necrotizing fasciitis-STSSa isolate20
        SS635T12 OF, emm12 Lancefield M type 12 reference strain, sof12 sfbX+27
        2108-98T12 OF, emm12 blood isolate, sof12 sfbX+This study
        3833-98T12 OF, emm12 blood isolate, sof12 sfbX+This study
        27-01T25 OF+, emm75 blood isolate, sof75 sfbX+This study
        140-01T28, OF+, emm87.3 blood isolate, sof87 sfbX+This study
        149-01T8/25/Imp19 OF+, emm92 isolate, sof92 sfbX+This study
        142-01T-NT OF+, emml 14 isolate from jointThis study
    GAS mutants
        NZ131:sofΔcatNZ131 with in-frame allelic replacement of sof by catThis study
        NZ131:sfbxΔcatNZ131 with in-frame allelic replacement of sfbX by catThis study
        NZ131:sof+sfbxΔcatNZ131 with in-frame allelic replacement of sof-sfbX by catThis study
    E. coli
        DH5αendA1 hsdR17(rKmK+) sup E44 thi-1 recA1 gyrA (Nalr) relA1 Δ(lacZYA-arg F)U169 [φ80dlac Δ(lacZ)M15]50
        MC1061F araD139 Δ(araABC-leu)7696 Δ(lac)X74 galU galK hsdR2 (rKmK+) mcrB1 rpsL (Strr)49
        Top 10F mcrA Δ(mrr-hsdRMS-mcrBC) φ80lacZΔM15 Δ(lac) X74 deoR recA1 araD139 Δ(ara-leu)7697 galU galK rpsL (Strr) endA1 nupG14
        BL21(DE3)F ompT hsdSB(rB mB) gal dcm (DE3)Novagen
    L. lactis
        NZ9000MG1363 (lacking nisin operon); pepN::nisRK26
Plasmids
    Chromosomal walking
        pBluescript IIColE1 ori Amp lacZαStratagene
    Allelic-exchange mutagenesis
        pCR2.1-TOPOColE ori Ampr Knr lacZα; T-A cloning vectorInvitrogen
        pHY304Temperature-sensitive pVE6007Δ derivative, Emr lacZα/MCSb of pBluescript37
        pACYC184rep(p15A) Cmr Tetr40
        pSof-TVpCR2.1 with 3,786-bp NZ131 DNA fragment containing sofThis study
        pSfbX-TVpCR2.1 with 2,589-bp NZ131 DNA fragment containing sfbXThis study
        pSof/SfbX-TVpCR2.1 with 5,947-bp NZ131 DNA fragment containing sof-sfbXThis study
        pSofΔcat-TVIn vivo PCR recombination derivative of pSof-TV with cat replacing sof gene precisely in frame, Ampr CmrThis study
        pSfbXΔcat-TVIn vivo PCR recombination derivative of pSfbX-TV with cat replacing sfbX gene precisely in frame, Ampr CmrThis study
        pSof/SfbXΔcat-TVIn vivo PCR recombination derivative of pSof/SfbX-TV with cat replacing sof-sfbX genes precisely in frame, Ampr CmrThis study
        pSofΔcat-KOpHY304 containing Δsof cat allele and flanking DNA, Emr CmrThis study
        pSfbXΔcat-KOpHY304 containing ΔsfbX cat allele and flanking DNA, Emr CmrThis study
        pSof/SfbXΔcat-KOpHY304 containing Δsof-sfbX cat allele and flanking DNA, Emr CmrThis study
    Complementation and heterologous expression
        pDC123E. coli-streptococcus shuttle vector, JS-3 replicon, phoS Cmr (cat)8
        pDCermpDCerm derivative with Ermr (erm of Tn916ΔE) replacing catThis study
        pAJI-sofpDCerm plus NZ131 sof gene behind vector and native promotersThis study
        pAJI-sfbXpDCerm plus NZ131 sfbX gene behind vector promotersThis study
    Expression of recombinant SfbX
        pCRT7/CT-TOPOColE ori, Ampr Zeor, T7 promoter, T-A cloning, V5 epitope, His6Invitrogen
        pStbX-V5-HisComplete sfbX ORF cloned in pCRT7/CT-TOPO for C-terminal tagThis study
  • a STSS, staphylococcal toxic shock syndrome.

  • b MCS, multiple cloning site.