TABLE 1.
Bacterial strains and plasmids
| Strain or plasmid | Description | Reference |
|---|---|---|
| Strains | ||
| GAS clinical isolates | ||
| NZ131 | T14 OF+, emm49 glomerulonephritis isolate | 45 |
| 5448 | T1 OF−, emm1.0 necrotizing fasciitis-STSSa isolate | 20 |
| SS635 | T12 OF−, emm12 Lancefield M type 12 reference strain, sof12 sfbX+ | 27 |
| 2108-98 | T12 OF−, emm12 blood isolate, sof12 sfbX+ | This study |
| 3833-98 | T12 OF−, emm12 blood isolate, sof12 sfbX+ | This study |
| 27-01 | T25 OF+, emm75 blood isolate, sof75 sfbX+ | This study |
| 140-01 | T28, OF+, emm87.3 blood isolate, sof87 sfbX+ | This study |
| 149-01 | T8/25/Imp19 OF+, emm92 isolate, sof92 sfbX+ | This study |
| 142-01 | T-NT OF+, emml 14 isolate from joint | This study |
| GAS mutants | ||
| NZ131:sofΔcat | NZ131 with in-frame allelic replacement of sof by cat | This study |
| NZ131:sfbxΔcat | NZ131 with in-frame allelic replacement of sfbX by cat | This study |
| NZ131:sof+sfbxΔcat | NZ131 with in-frame allelic replacement of sof-sfbX by cat | This study |
| E. coli | ||
| DH5α | endA1 hsdR17(rK−mK+) sup E44 thi-1 recA1 gyrA (Nalr) relA1 Δ(lacZYA-arg F)U169 [φ80dlac Δ(lacZ)M15] | 50 |
| MC1061 | F−araD139 Δ(araABC-leu)7696 Δ(lac)X74galU galK hsdR2 (rK−mK+) mcrB1 rpsL (Strr) | 49 |
| Top 10 | F−mcrA Δ(mrr-hsdRMS-mcrBC) φ80lacZΔM15 Δ(lac) X74 deoR recA1 araD139 Δ(ara-leu)7697 galU galK rpsL (Strr) endA1 nupG | 14 |
| BL21(DE3) | F−ompT hsdSB(rB− mB−) gal dcm (DE3) | Novagen |
| L. lactis | ||
| NZ9000 | MG1363 (lacking nisin operon); pepN::nisRK | 26 |
| Plasmids | ||
| Chromosomal walking | ||
| pBluescript II | ColE1 ori Amp lacZα | Stratagene |
| Allelic-exchange mutagenesis | ||
| pCR2.1-TOPO | ColE ori Ampr KnrlacZα; T-A cloning vector | Invitrogen |
| pHY304 | Temperature-sensitive pVE6007Δ derivative, EmrlacZα/MCSb of pBluescript | 37 |
| pACYC184 | rep(p15A) Cmr Tetr | 40 |
| pSof-TV | pCR2.1 with 3,786-bp NZ131 DNA fragment containing sof | This study |
| pSfbX-TV | pCR2.1 with 2,589-bp NZ131 DNA fragment containing sfbX | This study |
| pSof/SfbX-TV | pCR2.1 with 5,947-bp NZ131 DNA fragment containing sof-sfbX | This study |
| pSofΔcat-TV | In vivo PCR recombination derivative of pSof-TV with cat replacing sof gene precisely in frame, Ampr Cmr | This study |
| pSfbXΔcat-TV | In vivo PCR recombination derivative of pSfbX-TV with cat replacing sfbX gene precisely in frame, Ampr Cmr | This study |
| pSof/SfbXΔcat-TV | In vivo PCR recombination derivative of pSof/SfbX-TV with cat replacing sof-sfbX genes precisely in frame, Ampr Cmr | This study |
| pSofΔcat-KO | pHY304 containing Δsof cat allele and flanking DNA, Emr Cmr | This study |
| pSfbXΔcat-KO | pHY304 containing ΔsfbX cat allele and flanking DNA, Emr Cmr | This study |
| pSof/SfbXΔcat-KO | pHY304 containing Δsof-sfbX cat allele and flanking DNA, Emr Cmr | This study |
| Complementation and heterologous expression | ||
| pDC123 | E. coli-streptococcus shuttle vector, JS-3 replicon, phoS Cmr (cat) | 8 |
| pDCerm | pDCerm derivative with Ermr (erm of Tn916ΔE) replacing cat | This study |
| pAJI-sof | pDCerm plus NZ131 sof gene behind vector and native promoters | This study |
| pAJI-sfbX | pDCerm plus NZ131 sfbX gene behind vector promoters | This study |
| Expression of recombinant SfbX | ||
| pCRT7/CT-TOPO | ColE ori, Ampr Zeor, T7 promoter, T-A cloning, V5 epitope, His6 | Invitrogen |
| pStbX-V5-His | Complete sfbX ORF cloned in pCRT7/CT-TOPO for C-terminal tag | This study |
a
STSS, staphylococcal toxic shock syndrome.
b
MCS, multiple cloning site.