Table 1.

Strains and plasmids used in this study

Strain or plasmidRelevant genotype or phenotypeaReference or source
Strains
E. coli
  DH10BFφ80dlacZΔM15 recA1 Gibco, Gaithersburg, Md.
  BL21(DE3)F ompT gal [dcm] [lon] hsdSB (rB mB ), with DE3Novagen, Madison, Wis.
S. aureus
  RN4220NCTC8325-4 r m+ (restriction minus, modification plus)26
  BB255Essentially the same as NCTC83255
  8325-4NCTC8325, cured of known prophages34
  RN6390Derivative of NCTC8325 that maintains its hemolytic pattern when propagated on sheep erythrocytes35
  COL mec, high-Mcr clinical isolate, Tcr 25
  NewmanClinical isolate, high level of clumping factor (ATCC 25904)18
  IK181BB255 ΔrsbUVWsigB Emr 28
  IK183COL ΔrsbUVWsigB Emr 28
  IK184Newman ΔrsbUVWsigB Emr 28
  GP268BB255 (rsbUVWsigB)+-tetLTcr This study
  GP2698325-4 (rsbUVWsigB)+-tetLTcr This study
  MB25RN4220asp23 +(asp23P::luc+)-pEC-ermBEmr This study
  MB32Newmanasp23 +(asp23P::luc+)-pEC-ermBEmr This study
  MB33BB255asp23 +(asp23P::luc+)-pEC-ermBEmr This study
  MB49BB255 (rsbUVWsigB)+-tetL asp23 +(asp23P::luc+)-pEC-ermBTcr Emr This study
  MB61RN4220asp23 +(asp23P::luc+)-pBT-tetLTcr This study
  MB69Newman ΔrsbUVWsigB asp23 +(asp23P::luc+)-pBT-tetLTcr Emr This study
  MB90BB255 ΔrsbUVWsigB asp23 +(asp23P::luc+)-pBT-tetLTcr Emr This study
Plasmids
 pET-24b(+)Kmr; expression vectorNovagen
 pSP-luc+Apr; firefly luciferase casette vectorPromega
 pBC SK+Cmr; cloning vectorStratagene
 pAW8Tcr; pAMα1 origin andtetL gene of pHY300PLK, ColE1 originA. Wada, unpublished data
 pBTTcr; 1.6-kb PCR fragment oftetL gene of pHY300PLK into Alw26I-digested pBC SK(+)This study
 pEC1Apr; Emr; 1.45-kb ClaI ermB fragment of Tn551 in pUC189
 pIK6Apr; 6.6-kbPstI-EcoRI sigB fragment from strain 8325 in pUC1827
 pPG11Apr; Tcr; 252-bp MluI-BstXI fragment of rsbUfrom strain COL replacing the corresponding region of the 6.6-kbPstI-EcoRI sigB fragment from strain 8325 in pUC19This study
 pETasp23 510-bp PCR fragment of asp23 from strain 8325 in pET24bThis study
 pETrsbU COL 1-kb PCR fragment ofrsbU from strain COL in pET24bThis study
 pETsigB 770-bp PCR fragment of sigB from strain 8325 in pET24bThis study
 pSPasp23P 1.1-kb PCR fragment of asp23promoter from strain COL in pSP-luc+This study
 pECasp23P::luc+2.7-kbKpnI-EcoRIasp23P::luc+ fragment of pSPasp23P in pEC1This study
 pBTasp23P::luc+2.7-kbKpnI-EcoRIasp23P::luc+ fragment of pSPasp23P in pBTThis study
  • a Abbreviations are as follows: Apr, ampicillin resistant; Cmr, chloramphenicol resistant; Emr, erythromycin resistant; Kmr, kanamycin resistant; Mcr, methicillin resistant; Tcr, tetracyclin resistant.