TABLE 1.
Strains and plasmids used in this studya
| Strain or plasmid | Relevant property(ies)b | Reference or source |
|---|---|---|
| Strains | ||
| S. pneumoniae | ||
| D39 | CPS serotype 2 parent | 52 |
| AB1013 | D39 ΔspxB (allelic replacement of spxB with aphA-3); Kmr | Our laboratory (our unpublished data) |
| AG538 | D39 cps2B::pAG216; blue phenotype on X-gal; Cmr | This study |
| AG539 | Single-colony isolate of AG538; Cmr | This study |
| AG540 | pAG177 × AG539; repairs AG539 to parental Cps2B [the restored cps2B contains a same-sense mutation, cps2B3259T→C; D39(Cps2BD117D)] | This study |
| AG547 | pAG208 × AG539; cps2B3294G→A D39(Cps2BG129E) | This study |
| AG548 | pAG242 × AG539; cps2B2973G→A D39(Cps2BS22N) | This study |
| AG550 | pAG238 × AG539; cps2B2964G→A D39(Cps2BR19K) | This study |
| AG554 | pAG240 × AG539; D39Δcps2B | This study |
| AG557 | pAG255 × AG539; cps2B3314C→G,3315A→C D39(Cps2BH136A) | This study |
| AG562 | pAG269 × AG554; aliA::pAG269 aliA+ Emr; D39 Δcps2B vector control (ΔB/VC) | This study |
| AG563 | pAG271 × AG554; aliA::pAG271 aliA+ cps2B+ Emr; complemented D39 Δcps2B (ΔB/B+) | This study |
| AM1000 | D39 Δ(cps2A-cps2H); CPS− | 1 |
| E. coli MB053 | JM109 pMB053 Cps2B-His (Cps2B with an N-terminal His tag); Apr | 22 |
| Plasmids | ||
| pAG177 | pJY4164::EcoRI-digested product of Cps2–A24/Cps2–C34 PCR; contains cps2B; used to repair Δcps2B; recipient vector for mutant cps2B alleles (cps2B contains a same-sense mutation, cps2B3259T→C [D117D], as a result of a PCR error) | This study |
| pAG208 | pAG177 containing cps2B3294G→A (G129E) | This study |
| pAG216 | pEVP3 containing cps2B between SphI and BamHI sites | This study |
| pAG238 | pAG177 containing cps2B2964G→A (R19K) | This study |
| pAG240 | pJY4164::EcoRI-digested product of overlap extension PCR using primer sets Cps2–A24/Cps2–A43 and Cps2–C48/Cps2–C34 stored in pCR2.1 TOPO; used to generate Δcps2B | This study |
| pAG242 | pAG177 containing cps2B2973G→A (S22N) | This study |
| pAG255 | pJY4164::EcoRI-digested product of overlap extension PCR using primer sets Cps2–A24/Cps2–B28 and Cps2–B27/Cps2–C34; contains cps2B (H136A) with flanking sequences | This study |
| pAG269 | pJY4164::EcoRI/BamHI-digested product of primer set AliA-1/AliA-2 | This study |
| pAG271 | pAG269:: SpeI/SphI-digested product of primer set Cps2–B57/Cps2–B62; inserts cps2B after aliA; Cps2B+; Emr | This study |
| pEVP3 | Multiple-cloning site creates transcriptional fusions to downstream lacZ; Cmr | 53 |
| pJY4164 | Lacks S. pneumoniae origin of replication; Emr | 54 |
| pMB053 | pQE-41 encoding His-Cps2B | 22 |
| pQE-41 | IPTG-inducible expression vector; Apr | Qiagen |
a
See Table S1 in the supplemental material for additional strains and plasmids.
b
Superscript nucleotide numbers correspond to those reported under GenBank accession no. AF026471.3.