Table 1.

Strains and plasmids used in this study

Strain or plasmidReference(s) or sourceDescription
Strains
S. aureus
  RN6390 22,26 Laboratory strain that maintains its hemolytic pattern when propagated on sheep erythrocytes and has a genetic background similar to that of 8325-4
  RN4220 26 Mutant of 8325-4 that accepts foreign DNA
  COL 30 Methicillin-resistant S. aureus strain
  RUSA168 30 sigB mutant of COL (sigB::Tn551)
  ALC1001This study sigB mutant of RN6390
  ALC1497This studyALC1001 complemented with shuttle plasmid pALC1496 (with the sigB gene)
E. coli
  BL21NovagenHost strain for pET14b
  DU5384 27 Host strain carrying pBR322 with a 3-kb EcoRI-HindIII fragment of thehla gene
Plasmids
 pCR2.1InvitrogenPCR cloning vector
 pET14bNovagen E. coli expression vector
 pALC103 3 pSPT181 with a sarfragment from nucleotides 620 to 1349
 pALC1270This studypET14b with the sigB coding region cloned into theNdeI-BamHI sites
 pDG148 18 B. subtilis-E. colishuttle plasmid (8.2 kb) containing the pSpac promoter (IPTG inducible) followed by a polylinker site and lacI
 pSK236 19 S. aureus-E. colishuttle vector with pUC19 cloned into the HindIII site of pC194
 pALC1456This studypSK236 containing a 1.6-kb fragment derived from pDG148 and comprising, sequentially, the pSpac promoter, a polylinker site, and the lacI repressor
 pALC1496This studypALC1456 with the sigB gene (rbs + coding region) cloned into the polylinker site (SalI-PstI) derived from pDG148