Table 1.
Strains and plasmids used in this study
| Strain or plasmid | Propertiesa | Reference or source |
|---|---|---|
| S. pneumoniae | ||
| BX547 | D39 Δcps2K Cpsr; cps2E5544 G→T (D167Y) Kmr | 34 |
| BX552 | D39 Δcps2H Cps− cps2E5539 C→T (L200F); Kmr (in the original publication, this mutant was incorrectly identified as L199F) | 34 |
| BX605 | D39 Δcps2K Cpsr cps2E5632T→G (V196G) Kmr | 34 |
| BX612 | D39 Δcps2K Cpsr cps2E5539T→G (V165G) Kmr | 34 |
| D39 | Type 2 parent strain; Cps+ | 57 |
| DJ916 | D39 Δcps2F Cps− cps2E5718 A→G (H258R) Kmr | 25 |
| DJ918 | D39 Δcps2G Cpsr cps2E6059G→C (M338I) Kmr | 25 |
| DJ919 | D39 Δcps2G Cpsr cps2E6964C→A (R307S) Kmr | 25 |
| DJ921 | D39 Δcps2G Cpsr cps2E5529G→T (A162S) Kmr | 25 |
| JK901 | pJK101 X D39; Cps−; aphA-3 inserted into cps2E; Kmr | This study |
| JK903 | pBX146 X JK901; cps2E repair; Cps+ | This study |
| JK906 | pDJ238 X JK901; cps2E5632T→G (V196G) Cpsr | This study |
| JK907 | pDJ241 X JK901; cps2E5718A→G (H258R) Cpsr | This study |
| KA1522 | D39 Δcps2E Cps− | 24 |
| WU2 | Type 3 parent strain; Cps+ | 58 |
| E. coli | ||
| BL21-AI | F− ompT hsdSB(rB− mB−) gal dcm araB::T7 RNAP-tetA | Invitrogen |
| DB11 | met thi gal hasdR nal rif; plasmid-cured derivative of V854 from reference 59 | 59 |
| DH5α | F− Φ80lacZΔM15 Δ(lacZYA-argF) U169 recA1 endA1 hsdR17 (rK− mK+) phoA supE44 λ− thi-1 gyrA96 relA1 | Invitrogen |
| DJ005 | BL21-AI expressing Cps2I; Apr | 25 |
| DJ009 | BL21-AI expressing Cps2T; Apr | 25 |
| DJ011 | BL21-AI expressing Cps2G; Apr | 25 |
| DJ204 | BL21-AI expressing Cps2F, replaced GTG start with ATG; Apr | 25 |
| DJ238 | DB11 (pDJ248); used to transform cps2E5718A→G (H258R) | |
| DJ241 | DB11 (pDJ241); used to transform cps2E5632T→G (V196G) | |
| DJ252 | BL21-AI (pDJ252) expressing Cps2EH258R; Apr | This study |
| DJ253 | BL21-AI (pDJ253) expressing Cps2EA162S; Apr | This study |
| DJ254 | BL21-AI (pDJ254) expressing Cps2ED167Y; Apr | This study |
| DJ269 | BL21-AI (pDJ269) expressing Cps2EL200F; Apr | This study |
| DJ270 | BL21-AI (pDJ269) expressing Cps2EV196G; Apr | This study |
| DJ271 | DH5α (pDJ271); aphA-containing clone; Kmr | This study |
| JK101 | DB11 (pJK101); contains aphA-3-disrupted cps2E; Kmr | This study |
| KJ4152 | BL21-AI (pKJ4152) expressing full-length Cps2E; Apr | 25 |
| RC124 | BL21-AI (pET20b) vector control; Apr | 24 |
| Plasmids | ||
| pBX146 | pJY4164/cps2E; amplified from D39 using primer pair cps2-D3/cps2-T1; contains full-length parental cps2E. This plasmid is the same as the published pBX145. | 34 |
| pET20b | Protein expression vector; Apr | Novagen |
| pDJ238 | pBX146/cps2E5718A→G (H258R) amplified from DJ916 using primers E8/E11b | This study |
| pDJ241 | pBX146/cps2E5632T→G (V196G) amplified from BX605 using primers E8/E11b | This study |
| pDJ252 | pET-20b/cps2E5718 A→G (H258R) amplified from DJ916 using primers E8/E11b | This study |
| pDJ253 | pET-20b/cps2E5529G→T (A162S) amplified from DJ921 using primers E8/E11b | This study |
| pDJ254 | pET-20b/cps2E5544 G→T (D167Y) amplified from BX547 using primers E8/E11b | This study |
| pDJ269 | pET-20b/cps2E5539 C→T (L200F) amplified from BX552 using primers E8/E11b | This study |
| pDJ270 | pET-20b/cps2E5632 T→G (V196G) amplified from BX605 using primers E8/E11b | This study |
| pDJ271 | pGEM-T Easy/aphA-3-containing fragment amplified using primer pair DJ-03/DJ-04 | This study |
| pJK101 | pBX146/aphA-3 from BstBI-digested product of pDJ271 | This study |
| pJY4164 | S. pneumoniae suicide vector; Emr | 41 |
| pKJ4152 | pET-20b/cps2E amplified from S. pneumoniae D39 using primers E8/E11b | 25 |
a
cps2E superscripts indicate locations of mutations based on GenBank accession no. AF026471. Amino acid changes are indicated in parentheses. Cpsr, reduced capsule levels; →, nucleotide change; Apr, ampicillin resistant.
b
Primers are described in Materials and Methods.