TABLE 1.
Strains, plasmids, and primers used in this study
| Strain, plasmid, or primer | Relevant characteristics or sequence and gene | Source or reference |
|---|---|---|
| Strains | ||
| S. pneumoniae | ||
| TIGR4 | Wild-type, capsule type 4, transparent variant | 57 |
| JEN13 | TIGR4 psaA::Erm, transparent variant | This study |
| JEN15 | TIGR4 psaB::Erm | This study |
| JEN16 | JEN15 ΔpsaBC, transparent variant | This study |
| TIGR1001.1 | cps::Erm, capsule negative | This study |
| E. coli TOP10 | General cloning strain | Invitrogen |
| Plasmids | ||
| pCR2.1 | 3.9 kb, Ampr, ColE1 ori | Invitrogen |
| pGEM-T | 3.0 kb, Ampr, ColE1 ori | Promega |
| pJY4164 | 4.4 kb, Ermr, ColE1 ori | 66 |
| pJJ028 | pCR2.1 with an internal psaA fragment; Ampr | This study |
| pJJ034 | pJY4164 with an internal psaA fragment; Ermr | This study |
| pJJ043 | pGEM-T with fragment upstream of psaB; Ampr | This study |
| pJJ044 | pGEM-T with fragment downstream of psaC; Ampr | This study |
| pJJ045 | pJY4164 with fragment upstream of psaB; Ermr | This study |
| pJJ046 | pJJ045 with fragment downstream of psaC; Ermr | This study |
| pJJ052 | pCR2.1 with an internal psaB fragment; Ampr | This study |
| pJJ054 | pJY4164 1,410-bp fragment fusing partial psaB and psaC genes; Ermr | This study |
| Primersa | ||
| JWJ15 | GCAAGACCCACACGAATACGAAC; psaA | This study |
| JWJ16 | CAAGGTAGATAACATCAACGCCG; psaA | This study |
| JWJ20 | CGTGTCATCTTGTTCTCGC; psaB upstream | This study |
| JWJ35 | ggatccGGAGACACTGAGGTTTTCG; psaB upstream | This study |
| JWJ65 | ggtaccTGAAGAAGTCATCCAAAGGTAGGG; psaB upstream | This study |
| JWJ66 | aagcttGTAAACCAAGCATTGCCACC; psaC downstream | This study |
| JWJ75 | TTATCCCACATCAAGGTCAGGC; psaB | This study |
| JWJ76 | CACCAAACATCTGGCAATCAAGAC; psaB | This study |
| JWJ77 | CCTTTTCTTCTTTCTTAGCCCTCG; psaC | This study |
| JWJ79 | ggatccTTTATCGCTCCCAAACAAC; psaC downstream | This study |
| TT1 | GCCACTATCGACTACGCG |
a
Primers were based on the complete genome sequence of S. pneumoniae TIGR4 (57). Lowercase denotes mismatches used to create restriction endonuclease sites. All sequences are expressed 5′ to 3′.