Table 1 Strains and plasmids used in this study
Strain or plasmid | Description | Reference or source |
|---|---|---|
E. colistrains | ||
DH5α | F- φ80dlacZΔM15 Δ(lacZYA-argF)U169 deoR recA1 endA1 hsdR17(rk- mk+) phoA supE44 λ- thi-1 gyrA96 relA1 | Grant et al. [50] |
BL21 | F- ompT hsdSB(rB- mB-) gal dcm | Stratagene |
MS2066 | DH5α containing pSssFHis | This study |
MS2067 | BL21 containing pSssFHis | This study |
S. saprophyticus strains | ||
ATCC 15305 | Type strain (genome sequenced) | Kuroda et al. [8] |
MS1146 | Clinical isolate | AstraZeneca |
MS1146sssF | MS1146 isogenic sssF mutant | This study |
MS1146sssF(pSssF) | Complemented MS1146 sssF mutant | This study |
S. aureus strains | ||
SH1000 | Functional rsbU-repaired derivative of S. aureus 8325-4 | Horsburgh et al. [51] |
SH1000sasF | SH1000 isogenic sasF mutant | This study |
SH1000sasF(pSKSasF) | SH1000 sasF mutant complemented with sasF | This study |
SH1000sasF(pSKSssF) | SH1000 sasF mutant complemented with sssF | This study |
SH1000sasF(pSK5632) | SH1000 sasF mutant with empty pSK5632 vector | This study |
S. carnosus strains | ||
TM300 | Wild-type SK311 | Schleifer & Fischer [52] |
TM300(pSssF) | TM300 containing pSssF | This study |
Plasmids | ||
pBAD/HisB | Cloning and protein expression vector, containing N-terminal 6 × His tag; Apr | Invitrogen |
pNL9164 | E. coli/S. aureus TargeTron shuttle vector (temperature sensitive); Apr Emr | Sigma |
pSK5632 | Cloning and expression E. coli/S. aureus shuttle vector; Apr Cmr | Grkovic et al. [53] |
pPS44 | Staphylococcal vector, contains replicon and cat gene of pC194; Cmr | Wieland [54] |
pSssFHis | 1330 bp MS1146 sssF fragment, amplified with primers 873 and 874, digested with EcoRI/XhoI and cloned into EcoRI/XhoI-digested pBAD/HisB, with in-frame N-terminal 6 × His tag; Apr | This study |
pNK24 | pNL9164 shuttle vector retargeted with primers 1001-1003, EBSU to knock out MS1146 sssF (TargeTron system); Apr Emr | This study |
pNK41 | pNL9164 shuttle vector retargeted with primers 2065-2067, EBSU to knock out SH1000 sasF (TargeTron system); Apr Emr | This study |
pSKSssF | 2394 bp fragment, including entire sssF gene from MS1146, amplified with primers 839 and 840 and cloned into the BamHI site of pSK5632; Apr Cmr | This study |
pSssF | 2400 bp BamHI/XbaI fragment, containing sssF gene, subcloned from pSKSssF into BamHI/XbaI-digested pPS44; Cmr | This study |
pSKSasF | 2175 bp fragment, including sasF gene from S. aureus NCTC 8325, amplified with primers 2084 and 2085 and cloned into the HindIII site of pSK5632; Apr Cmr | This study |