TABLE 1.
Strains and plasmids used in this study
| Strain(s) or plasmid(s) | Propertiesa | Reference or source |
|---|---|---|
| S. pneumoniae strains | ||
| AM1000 | Δ[cps2A to cps2H]; type 2 Cps− | 39 |
| BX505 | pBX110 × D39, Δcps2H, Cps−; cps2E5765delT (L244* premature stop) | This study |
| BX511 | pBX113 × D39, Δcps2K, Cpsr; cps2E5953G→T (G303V) | This study |
| BX512 | pBX113 × D39, Δcps2K, Cpsr; cps2E5920G→A (G292R) | This study |
| BX513 | pBX116 × BX511, cps3D repair of Δcps2K, Cpsr | This study |
| BX515 | pBX115 × BX511, cps2K repair, Cpsr | This study |
| BX516 | pJD377 × D39, Em marker insertion downstream of capsule locus, Cps+ | This study |
| BX518 | pBX115 × BX512, cps2K repair, Cpsr | This study |
| BX519 | BX516 × BX511, Emr, capsule replacement in BX511, Cps+ | This study |
| BX522 | BX516 × BX512, Emr; capsule replacement in BX511, Cps+ | |
| BX532, BX539 | pBX113 × D39, independent Δcps2K derivatives; Cpsr; cps2E6132C→A (P363T) | This study |
| BX533 | pBX113 × D39, Δcps2K, Cpsr; cps2E6319A→G (D425G) | This study |
| BX535 | pBX123 × D39, Em insertion upstream of capsule locus promoter, Cps+ | This study |
| BX540 | pBX115 × BX533, cps2K repair, Cpsr | This study |
| BX544 | pBX145 × BX518, cps2E repair in BX518, Cps+ | This study |
| BX545 | pBX145 × BX515, cps2E repair in BX515, Cps+ | This study |
| BX547, BX548, BX549 | pBX113 × D39, Δcps2K, Cpsr; independent derivatives with respective cps2E mutations 5544G→T (D167Y), 6350G→T (W435C), and 6015delA (L369* premature stop) | This study |
| BX550 | pBX113 × D39, Δcps2K, Cpsr; unknown suppressor mutation | This study |
| BX551 | pBX113 × D39, Δcps2K, Cpsr; A-to-G transition 4 bp downstream of −10 sequence of capsule promoter | This study |
| BX552 | pBX110 × D39, Δcps2H, Cps−; cps2E5539C→T (L199F) | This study |
| BX554, BX555 | pBX113 × D39, Δcps2K, Cpsr; independent derivatives with respective cps2E mutations 6198A→G (E385G) and 6201T→C (Y386H) | This study |
| BX556 | pBX113 × D39, Δcps2K, Cpsr; 1-kb vector insertion in cps2L region | This study |
| BX605-BX607, BX609-BX612 | pBX113 × D39, Δcps2K, Cpsr; independent derivatives with respective cps2E mutations 5632T→G (V196G), 5607insT (E191* premature stop), 6178C→G (T378R), 5984G→C (K312N), 6178C→G (T378R), 6349G→A (W407*stop), and 5539T→G (V165G) | This study |
| BX635 | pBX113 × D39, Δcps2K, Cpsr; cps2E6060T→C (F339L) | This study |
| BX667-BX669 | pBX190 × D39, Δcps2J, Cps−; independent derivatives with respective cps2E mutations 6276G→A (G411R), 5839T→G (I265S), and 6157G→C (G371A) | This study |
| D39 | Type 2 parent strain, Cps+ | 5 |
| KA1521 | Δcps2E, type 2, Cps− | 15 |
| WU2 | Type 3 parent strain, Cps+ | 11 |
| E. coli strains | ||
| BX163 | M15(pREP4, pBX163) | This study |
| BX165 | M15(pREP4, pBX165) | This study |
| DH5αF′ | F′ φ80lacZΔM15 Δ(lacZYA-argF)U169 deoR recA1 endA1 hsdR17(rK− mK+) phoA supE44 λ−thi-1 gyrA96 relA1 | Life Technologies, Inc. |
| TOP10 | F−mcrA Δ(mrr-hsdRMS-mcrBC) φ80lacZΔM15 ΔlacX74 recA1 deoR araD139 Δ(ara-leu)7697 galU galK rpsL (Strr) endA1 nupG | Invitrogen |
| M15(pREP4) | F− Nals Strs Rifs Thi− Lac− Ara+ Gal+ Mtl− RecA+ Uvr+ Lon+ | QIAGEN |
| Plasmids | ||
| pCR 2.1 TOPO | PCR cloning vector; Apr Kmr | Invitrogen |
| pJY4164 | S. pneumoniae suicide vector; Emr | 72 |
| pJD377 | pJY4164::type 3 plpA/tnpA region | 23 |
| pBX105 | pJY4164 plus PCR fragments from primer pairs Cps2-G0/Cps2-G2 and Cps2-I2/Cps2-I3, for cps2H deletion | This study |
| pBX108 | pJY4164 plus PCR fragments from primer pairs Cps2-J12303/Cps2-J2 and Cps2-P1/Cps2-P15468, for cps2K deletion | This study |
| pBX110 | Km resistance gene, aphA-3, between two fragments of pBX105 | This study |
| pBX113 | Km resistance gene, aphA-3, between two fragments of pBX108 | This study |
| pBX115, pBX121 | pJY4164 plus PCR fragment from primer pair Cps2-J1/Cps2-P2, for repair of cps2K deletion | This study |
| pBX116 | pJY4164 plus PCR fragment from primer pair Cps3D-F/Cps3D-R, for cps3D repair of cps2K deletion | This study |
| pBX123 | pJY4164 plus 900-bp fragment excised with EcoRI from pCV646 | This study |
| pBX145 | pJY4164 plus PCR fragment from primer pair Cps2-D3/Cps2-T1, for repair of cps2E | This study |
| pBX163 | pQE-40 containing the full-length cps2K lacking GTG start; from primer pair Cps2KORF-BglII/Cps2KORF-KpnI | This study |
| pBX165 | pQE-40 lacking dihydrofolate reductase region (BamHI-KpnI, deletion) | This study |
| pBX190 | pJY4164 plus PCR fragments from primer pairs Cps2-I4/Cps2-J8 and Cps2-J7/Cps2-K1, for deletion of cps2K; aphA-3 excised from pBX113 and inserted between two fragments | This study |
| pCV646 | pGEM cloning vector plus PCR fragment from c-ups1/c-ups2; Apr | This study |
| pQE40 | Expression vector, N-terminal His6 tag; Kmr | QIAGEN |
| pSF151 | Streptococcal shuttle vector containing the Km resistance gene, aphA-3 | 56 |
a
Cpsr, reduced capsule levels. cps2E superscripts indicate mutations and their locations based on GenBank accession no. AF026471. Amino acid changes are indicated in parentheses. del, deletion; ins, insertion; →, nucleotide change.