TABLE 1.
Strains, plasmids and primers used in this study
| Strain, plasmid, or primer | Relevant characteristics | Source or reference |
|---|---|---|
| S. aureus | ||
| RN4220 | rK− mK+ | 24 |
| MN8 | tstH+; clinical isolate from nonmenstrual toxic shock syndrome case | 41 |
| AMD283 | MN8 agrD | This study |
| Plasmids | ||
| LITMUS28 | Cloning vector | New England Biolabs |
| pCR2.1-TOPO | TA cloning vector | Invitrogen |
| pKEN-GFPmut2 | gfpmut2 | 10 |
| pRSET10-B | yfp10B | R. Heim |
| pE194 | ermC | 19 |
| pCE104 | Shuttle vector containing pE194 and pUC18; Emr | 33 |
| pCE107 | pCE104 containing HindIII-SalI MN8 tstH chromosomal fragment; Emr | P. Schlievert |
| pKSV7 | pUC18-pC194 chimera; temperature sensitive; Cmr | 45 |
| pDB1 | PCR-amplified (O1 and O2) agr region from RN4220 cloned into XhoI-HindIII-digested LITMUS28 | This study |
| pDB2 | BamHI-PstI gfpmut2 from pKEN cloned into BamHI-PstI-digested LITMUS28 | This study |
| pDB3 | PCR-amplified ermC from pE194 cloned into EcoRI- and BglII-digested LITMUS28 | This study |
| pDB4 | BglIIEcoRI ermC fragment from pDB3 cloned into BglII- and EcoRI-digested pDB2 | This study |
| pDB6 | pUC18 containing XhoI (SalI)-HindIII agr fragment from pDB1 | This study |
| pDB9 | pDB6 with BglII site introduced into the RNAIII region using O35, O36 | This study |
| pDB12 | BamHI-BglII gfpmut2ermC fragment from pDB4 cloned into BglII-digested pDB9 | This study |
| pDB13 | BamHI-HindIII agr P3-gfpmut2ermC fragment from pDB12 cloned into BamHI- and HindIII-digested pCE107 | This study |
| pDB22 | agr P3-gfpmut2 reporter; PCR-amplified (O1 and O62) agr P3-gfpmut2ermC fragment from pDB13 cloned into BamHI-HindIII-digested pCE107 | This study |
| pDB39 | pUC18/pC194 shuttle vector containing BamHI-BglII yfp10B fragment from pDB44 | This study |
| pDB44 | pCR2.1-TOPO containing yfp10B PCR amplified from BamHI-digested pRSET10-B, yfp10B contains gene10 ribosome-binding site | This study |
| pDB52 | BamHI-BglII fragment (containing ermC) of pDB3 cloned into BamHI-digested pKSV7 | This study |
| pDB53 | agrCA fragment PCR amplified from MN8 (O63 and O68) and cloned into XbaI-digested pDB52 | This study |
| pDB59 | PCR-amplified (O110 and O111) agr P2-P3 region from MN8 cloned into BamHI-digested pDB39; agr P3-yfp10B; Cmr | This study |
| pDB60 | agrB-P3 fragment PCR amplified (O112 and O113) from MN8 and cloned into BamHI- and BglII-digested pDB53 | This study |
| pDB63 | PCR-amplified (O126 and O127) sar P1 region from MN8 cloned into BamHI-digested pDB39; Cmr | This study |
| pJY202 | agr P3-yfp10B reporter; pCE104 containing agr P3-yfp10BKpnI-HindIII fragment of pDB59; Emr | This study |
| pJY209 | sar P1-yfp10B reporter; pCE104 containing sar P1-yfp10BKpnI-HindIII fragment of pDB63; Emr | This study |
| Primer (5′-3′) | ||
| O1 | TATATAAGCTTGAGCTTGGGAGGGGCTCACGACC | |
| O2 | TATATCTCGAGAGCTTGCTCAAGCACCTCATAAGG | |
| O20 | ATGATCGAATTCTTATTTGTACAATTCA | |
| O35 | GCTTTTAGCATGTTTTAATATAACTAGATCTCAGAGATGTGATGGAAAATAG | |
| O36 | CTATTTTCCATCACATCTCTGAGATCTAGTTATATTAAAACATGCTAAAAGC | |
| O62 | ATAATGGATCCATTTTAACATAAAAAAATTTACAGTTAAG | |
| O63 | GCACGTCTAGAGGTATACAATGAAACGTTGTATCG | |
| O68 | ATACTCTCTAGACTATAAGAGAAAGTGTGATAG | |
| O93 | ATGGGAAGCTTCGTGGATCCTCTAGATTTAAG | |
| O94 | GTTCTTCTCCTTTACTCATATGTATATCTCC | |
| O97 | CGCTGGCAGATCTTTATTATTTGTATAGTTCATCCATGCC | |
| O110 | GGATCCACCACTCTCCTCACTGTTATTATACGA | |
| O111 | AGATCTTTTTCCATCACATCTCTGTGATCTAGT | |
| O112 | TAGATCTATCAAGGATGTGATGTTATGAAAGT | |
| O113 | AGGATCCTTTTTCATAATTTAGTCCTCCTTTGAA | |
| O126 | GGATCCGCTGATATTTTTGACTAAACCAAATG | |
| O127 | GAGATCTCCTCCCTATTTGATGCATCTTG |