TABLE 1.
Bacterial strains and plasmids used in this study
| Strain or plasmid | Genotype (description)a | Antibiotic resistanceb | Reference or source |
|---|---|---|---|
| Bacterial strains | |||
| S. pneumoniae | |||
| EL59 | R6 (unencapsulated laboratory strain R6 derived from intermediates of strain D39) | None | 25 |
| IU1473 | R6 ΔvicR<>ermAM kan-t1t2-Pc-pcsB | Kanr | 31 |
| IU1690 | D39 (single-colony isolate of serotype 2 strain encapsulated D39 NCTC 7466) | None | 25 |
| IU1781 | D39 rpsL1 | Strr | 23 |
| IU1824 | D39 rpsL1 Δcps2A′-cps2H′ | Strr | 25 |
| IU1876 | R6 ftsZ-(C)-FLAG3 intergenic::Pc-ermAM (EL59 transformed with fusion amplicon) | Ermr | This study |
| IU1885 | D39 rpsL1 ΔwalKSpn::Pc-[Kanr-rpsL+] | Strs Kanr | 19 |
| IU1888 | D39 rpsL1 ΔwalJSpn::Pc-[Kanr-rpsL+] (IU1781 transformed with fusion amplicon) | Strs Kanr | This study |
| IU1896 | D39 rpsL1 ΔwalKSpn | Strr | 19 |
| IU3126 | R6 pcsB-(C)-L-FLAG3 intergenic::Pc-ermAM (EL59 transformed with fusion amplicon) | Ermr | This study |
| IU3150 | D39 ΔbgaA′::Pc-kant1t2-Pfcsk-pcsB (IU1690 transformed with fusion amplicon) | Kanr | This study |
| IU3348 | R6 ΔbgaA′::Pc-kant1t2-Pfcsk-pcsB-(C)-L-FLAG3 (EL59 transformed with fusion amplicon) | Kanr | This study |
| IU3477 | R6 ΔbgaA′::Pc-kant1t2-Pfcsk-walJSpn-(C)-L-FLAG3 (EL59 transformed with fusion amplicon) | Kanr | This study |
| IU3479 | R6 ΔbgaA′::Pc-kant1t2-Pfcsk-walRSpn-(C)-L-FLAG3 (EL59 transformed with fusion amplicon) | Kanr | This study |
| IU3689 | D39 Δcps2A′-cps2H′ rpsL1 walJSpn+ intergenic::Pc-[Kanr-rpsL+] (IU1824 transformed with fusion amplicon) | Strs Kanr | This study |
| IU3816 | D39 Δcps2A′-cps2H′ rpsL1 walJSpn-(C)-L-FLAG3 (IU3689 transformed with fusion amplicon) | Strr | This study |
| IU3953 | D39 Δcps2A′-cps2H′ rpsL1 ΔwalJSpn (IU3689 transformed with fusion amplicon) | Strr | This study |
| IU4021 | D39 Δcps2A′-cps2H′ rpsL1 ΔwalKSpn::Pc-[Kanr-rpsL+] (IU1824 transformed with ΔwalKSpn::Pc-[Kanr-rpsL+] amplicon from IU1885) | Kanr | This study |
| IU4052 | D39 Δcps2A′-cps2H′ rpsL1 walRSpn-(C)-L-FLAG3 (IU4021 transformed with fusion amplicon) | Strr | This study |
| IU4062 | D39 Δcps2A′-cps2H′ rpsL1 walKSpn-(C)-FLAG (IU4021 transformed with fusion amplicon) | Strr | This study |
| IU4092 | D39 Δcps2A′-cps2H′ rpsL1 ΔwalKSpn::Pc-[Kanr-rpsL+] walJSpn-(C)-L-FLAG3 (IU3816 transformed with ΔwalKSpn::Pc-[Kanr-rpsL+] from IU1885) | Strs Kanr | This study |
| IU4099 | D39 Δcps2A′-cps2H′ rpsL1 ΔwalKSpn walJSpn-(C)-L-FLAG3 (IU4092 transformed with ΔwalKSpn amplicon from IU1896) | Strr | This study |
| E. coli | |||
| EL27 | BL21(DE3)(pLysS)(pSP001) | Ampr Cmr | 31 |
| IU4019 | BL21(DE3) Rosetta(pLysS)(pIU251) | Kanr Cmr | This study |
| IU4223 | BL21(DE3) Rosetta(pLysS)(pIU259) | Kanr Cmr | This study |
| Plasmids (E. coli) | |||
| pSP001 | pET16b [expression of (N)-His10-WalRSpn] | Ampr Cmr | 31 |
| pIU251 | pSumo [expression of (N)-Sumo-WalKSpn ΔN35-(C)-FLAG, where ΔN35 removed the transmembrane domain of WalKSpn] | Kanr Cmr | This study |
| pIU259 | pET28a [expression of WalJSpn-(C)-L-FLAG3] | Kanr Cmr | This study |
Primers used to synthesize fusion amplicons are listed in Table S1 in the supplemental material. (N)- or (C)- indicates that fusions were made to the amino- or carboxyl-terminal ends of reading frames, respectively. L refers to a 10-amino-acid spacer linker (GSAGSAAGSG) based on reference 42. FLAG3 indicates three tandem copies of the FLAG epitope (DYKDDDDK) (20). Intergenic means that the resistance gene was cloned into the intergenic region following the indicated gene. BL21(DE3)(pLysS), BL21(DE3) Rosetta(pLysS), pET16b, and pET28a were purchased from Novagen, and pSumo was purchased from LifeSensors.
Antibiotic resistance markers: Kanr, kanamycin; Strr, streptomycin, Ermr, erythromycin; Ampr, ampicillin; Cmr, chloramphenicol. Concentrations of antibiotics used for S. pneumoniae strains: 250 μg Str per ml (Sigma S6501), 250 μg Kan per ml (Sigma K0254), and 0.3 μg Erm per ml (Sigma E6376); for E. coli strains, 34 μg Cm per ml (Sigma C0378) and 30 μg Kan per ml and 100 μg Amp per ml (Sigma A5354).