Table 1.
Bacterial strains used in this study
| Strain | Relevant featuresa | Reference or source |
|---|---|---|
| Escherichia coli strains | ||
| XL1 Blue | Cloning strain; Tetr; ANG127 | Stratagene |
| CLG190 | Cloning strain which reduces plasmid copy number; Tetr; ANG1141 | Dana Boyd; 20 |
| ANG126 | pTS1 in DH5α; vector with temperature-sensitive replication in S. aureus; Ampr | 34 |
| ANG201 | pCN34 in E. coli; E. coli-S. aureus shuttle vector; Kanr Ampr | 12 |
| ANG243 | pCL55 in XL1 Blue; S. aureus single-site integration vector; Ampr | 30 |
| ANG284 | pitet in XL1 Blue; pCL55 containing Atet-inducible promoter; Ampr | 21 |
| ANG374 | pCL55-ypfP/ltaA in XL1 Blue; Ampr | 21 |
| ANG474 | pMutin-HA in E. coli; Ampr | Bacillus Genetic Stock Center |
| ANG483 | pOK-ltaS in XL1 Blue; Kanr | 22 |
| ANG503 | pCL55-ltaS in XL1 Blue; Ampr | 45 |
| ANG505 | pCL55-yflE in CLG190; Ampr | This study |
| ANG584 | pitet-ltaS-his in XL1 Blue; ltaS with C-terminal His tag under Atet-inducible promoter control; Ampr | This study |
| ANG590 | pitet-sec-eltaS in XL1 Blue; protein A signal peptide fused to eltaS domain under Atet-inducible promoter control; Ampr | This study |
| ANG1112 | pitet-ltaS-T300A in XL1 Blue; ltaS(T300A) allele under Atet-inducible promoter control; Ampr | 32 |
| ANG1211 | pitet-TMsrtA-eltaS in XL1 Blue; sortase TM fused to eltaS domain under Atet-inducible promoter control; Ampr | This study |
| ANG1212 | pitet-1TM-eltaS in XL1 Blue; first TM of ltaS fused to linker plus eltaS domain under Atet-inducible promoter control; Ampr | This study |
| ANG1213 | pitet-3TM-eltaS in XL1 Blue; first three TM of ltaS fused to linker plus eltaS domain under Atet-inducible promoter control; Ampr | This study |
| ANG1214 | pitet-5TMltaA-eltaS in XL1 Blue; first five TM of ltaA fused to linker plus eltaS domain under Atet-inducible promoter control; Ampr | This study |
| ANG1221 | pCN34-5TM in XL1 Blue; 5TM of ltaS under native promoter control; Kanr Ampr | This study |
| ANG1222 | pCN34-ltaS in XL1 Blue; ltaS under native promoter control; Kanr Ampr | This study |
| ANG1242 | pOK-ltaSS218P in XL1 Blue; ltaS(S218P) allele under native promoter control; Kanr | This study |
| ANG1244 | pitet-ltaSS218P in XL1 Blue; ltaS(S218P) allele under Atet-inducible promoter control; Ampr | This study |
| ANG1336 | pitet-ltaSTM-eyflE-his in CLG190; 5TM of ltaS fused to linker plus eyflE domain with C-terminal His tag under Atet-inducible promoter control; Ampr | This study |
| ANG1337 | pitet-yflETM-eltaS in XL1 Blue; 5TM of yflE fused to linker plus eltaS domain under Atet-inducible promoter control; Ampr | This study |
| ANG1368 | pitet-ltaSS218P-his in XL1 Blue; ltaS(S218P) allele with C-terminal His tag under Atet-inducible promoter control; Ampr | This study |
| ANG1577 | pALC2073 in XL1 Blue; Ampr; ANG1577 | 5 |
| ANG1631 | pCN34itetH in XL1 Blue; pCN34 containing Atet-inducible promoter from pALC2073; Kanr Ampr | This study |
| ANG1689 | pCN34itet-ltaST300A in XL1 Blue; ltaS(T300A) allele under Atet-inducible promoter control; Kanr Ampr | This study |
| ANG1811 | pCN34itetH-spsB in XL1 Blue; spsB under Atet-inducible promoter control; Kanr Ampr | This study |
| ANG1815 | pTS1-ΔspsB::erm in XL1 Blue; Ampr | This study |
| ANG1963 | pCN34itetH-spsB-T114C in XL1 Blue; spsB with point mutation T114C under Atet-inducible promoter control; Kanr Ampr | This study |
| ANG1964 | pMutinHA-spsB-T114C in XL1 Blue; spsB with point mutation T114C under IPTG-inducible promoter control; Ampr | This study |
| ANG2007 | pCL55spac-spsB-T114C in XL1 Blue; spsB with point mutation T114C under IPTG-inducible promoter control; Kanr Ampr | This study |
| ANG2158 | pCN34itet-sec-eltaS in XL1 Blue; protein A signal peptide fused to eltaS domain under Atet-inducible promoter control; Kanr Ampr | This study |
| ANG2159 | pCN34itet-TMsrtA-eltaS in XL1 Blue; sortase TM fused to eltaS domain under Atet-inducible promoter control; Kanr Ampr | This study |
| ANG2160 | pCN34itet-1TM-eltaS in XL1 Blue; first TM of ltaS fused to linker plus eltaS domain under Atet-inducible promoter control; Kanr Ampr | This study |
| ANG2161 | pCN34itet-3TM-eltaS in XL1 Blue; first three TM of ltaS fused to linker plus eltaS domain under Atet-inducible promoter control; Kanr Ampr | This study |
| ANG2162 | pCN34itet-5TMltaA-eltaS in XL1 Blue; first five TM of ltaA fused to linker plus eltaS domain under Atet-inducible promoter control; Kanr Ampr | This study |
| ANG2163 | pCN34itet-ltaSTM-eyflE-his in XL1 Blue; 5TM of ltaS fused to linker plus eyflE domain containing C-terminal His tag under Atet-inducible promoter control; Kanr Ampr | This study |
| ANG2164 | pCN34itet-yflETM-eltaS in XL1 Blue; 5TM of yflE fused to linker plus eltaS domain under Atet-inducible promoter control; Kanr Ampr | This study |
| Staphylococcus aureus strains | ||
| RN4220 | Transformable laboratory strain; ANG113 | 29 |
| SEJ1 | RN4220 Δspa; ANG314 | 21 |
| ANG499 | RN4220 itaS; strain with IPTG-inducible ltaS expression; Ermr; IPTG added | 22 |
| ANG514 | pitet-ltaS integrated into strain ANG499; Ermr Camr; IPTG added | 22 |
| ANG513 | pitet integrated into strain ANG499; Ermr Camr; IPTG added | 22 |
| ANG587 | pitet-ltaS-his integrated into strain ANG499; Ermr Camr; IPTG added | This study |
| ANG595 | pitet-sec-eltaS integrated into strain ANG499; Ermr Camr; IPTG added | This study |
| ANG1130 | ANG499 with pCN34; Ermr Kanr; IPTG added | 45 |
| ANG1217 | pitet-TMsrtA-eltaS integrated into strain ANG499; Ermr Camr; IPTG added | This study |
| ANG1218 | pitet-1TM-eltaS integrated into strain ANG499; Ermr Camr; IPTG added | This study |
| ANG1219 | pitet-3TM-eltaS integrated into strain ANG499; Ermr Camr; IPTG added | This study |
| ANG1220 | pitet-5TMltaA-eltaS integrated into strain ANG499; Ermr Camr; IPTG added | This study |
| ANG1226 | ANG595 with pCN34; Ermr Camr Kanr; IPTG added | This study |
| ANG1227 | ANG595 with pCN34-5TM; Ermr Camr Kanr; IPTG added | This study |
| ANG1228 | ANG595 with pCN34-ltaS; Ermr Camr Kanr; IPTG added | This study |
| ANG1246 | pitet-ltaSS218P integrated into strain ANG499; Ermr Camr; IPTG added | This study |
| ANG1346 | pitet-ltaSTM-eyflE-his integrated into strain ANG499; Ermr Camr; IPTG added | This study |
| ANG1347 | pitet-yflETM-eltaS integrated into strain ANG499; Ermr Camr; IPTG added | This study |
| ANG1370 | pitet-ltaSS218P-his integrated into strain ANG499; Ermr Camr; IPTG added | This study |
| ANG1571 | ANG499 with pCN34itet-ltaS; Ermr Kanr; IPTG added | 45 |
| ANG1658 | ANG499 with pCN34itet-yvgJ; Ermr Kanr; IPTG added | 45 |
| ANG1690 | ANG595 with pCN34itet-ltaST300A; Ermr Kanr; IPTG added | This study |
| ANG1755 | SEJ1 Δsbi | Lab strain collection |
| ANG1786 | 4S5; SEJ1 ΔltaS; LTA-negative suppressor strain that can grow without LTA | R. Corrigan |
| ANG1816 | SEJ1 with pCN34itet-spsB; Kanr | This study |
| ANG1820 | SEJ1 spsB::erm with pCN34itet-spsB; Ermr Kanr | This study |
| ANG2008 | SEJ1 Pspac-spsB; pCL55spac-spsB-T114C integrated into strain SEJ1; Camr | This study |
| ANG2009 | SEJ1 spsB::erm Pspac-spsB; spsB::erm transduced from strain ANG1820 into strain ANG2008; Ermr Camr; IPTG added | This study |
| ANG2165 | ANG499 with pCN34itet-sec-eltaS; Ermr Kanr; IPTG added | This study |
| ANG2166 | ANG499 with pCN34itet-TMsrtA-eltaS; Ermr Kanr; IPTG added | This study |
| ANG2167 | ANG499 with pCN34itet-1TM-eltaS; Ermr Kanr; IPTG added | This study |
| ANG2168 | ANG499 with pCN34itet-3TM-eltaS; Ermr Kanr; IPTG added | This study |
| ANG2169 | ANG499 with pCN34itet-5TMltaA-eltaS; Ermr Kanr; IPTG added | This study |
| ANG2170 | ANG499 with pCN34itet-ltaSTM-eyflE-his; Ermr Kanr; IPTG added | This study |
| ANG2171 | ANG499 with pCN34itet-yflETM-eltaS; Ermr Kanr; IPTG added | This study |
| RN6390 | RN6390; virulent laboratory strain derived from NCTC 8325; ANG2153 | 36 |
| RN6390 htrA1htrA2 | RN6390 htrA1htrA2; Camr Specr; ANG1649 | 40 |
| AH1263 | LAC*; Erm-sensitive community-acquired methicillin-resistant S. aureus LAC strain; ANG1575 | 9 |
| AH1919 | LAC* Δaur ΔsspAB ΔscpA spl::erm (LAC* protease KO); ANG2038 | This study |
| Bacillus subtilis strain | ||
| 168 | Transformable lab strain; trpC2; ANG1691 | 11 |
a
Antibiotics were used at the following concentrations: for E. coli cultures, ampicillin (Amp) at 100 μg/ml and kanamycin (Kan) at 30 μg/ml; for S. aureus cultures, erythromycin (Erm) at 10 μg/ml, chloramphenicol (Cam) at 7.5 to 10 μg/ml, kanamycin (Kan) at 90 μg/ml, spectinomycin (Spec) at 100 μg/ml, and IPTG at 1 mM. Note that an Atet-inducible promoter was used in S. aureus.