TABLE 1.

Bacterial strains and plasmids

Strain or plasmidGenotype and/or phenotypeaReference or source
Strains
    E. coli
        MC1061F′ araD139 Δ(ara-leu)7696 Δ(lac)X74 galU galK hsdR2(rK mK+) mcrB1 rpsL (Strr) 45
        DH5αF′ endA (rK mK+) supE44 thi-1 hsdR17 gyrA (Nalr) recA1 Δ(lacZYA-orgF)U169 dcoR relA1 φ80Δlac(lacZ)M15 50
        XL1-BlueF′::Tn10 proA+B+ lacIq Δ(lacZ)M15 recA1 endA1 gyrA96 (Nalr) thi-1 hsdR17(rK mK+) glnV44 supE44 relA1 lacStratagene
    S. agalactiae
        COH1Wild type; serotype III; Tetr 28
        COH1-11COH1; neuA::Tn916ΔE asialo-CPS -Tetr Ermr 47
        COH1-13COH1; cpsE::Tn916ΔE CPS Tetr Ermr 35
        COH1-350COH1; ΔcpsK Tetr CmrThis study
        COH1-350(pLM104)COH1; ΔcpsK pBL26 cpsK recombinant plasmid; Tetr Cmr CPS+This study
        COH1-355COH1; cpsL::km-Ω-2 Tetr KmrThis study
    S. pneumoniae
        NCTC11902Wild type; serotype 14 27
Plasmids
    pBlueScript II KS(+)f1(+) ori, ColE1 ori; lacZα Apr; 3.0 kbStratagene
    pGEM T-Easyf1(+) ori, ColE1 ori; lacZα Apr; 3.0 kbPromega
    pLZ12spec3.7 kb; Spr 22
    pBS9.0 cpsIJKL neuBCDA; pBS derivative; Apr; 11.8 kb 9
    pDC143 cpsJKL neuB; pBlueScript II KS(+) with 5.4-kb EcoRI/NsiI fragment of pBS9.0; Apr; 8.3 kbThis study
    pDC123 phoZ cat; gram-positive/negative blue-white screening expression vector; Cmr; 4.5 kb 9
    pDC147 cpsJ cat cpsL neuB ΔcpsK; pDC143 with exact allelic exchange cpsK replacement with cat; Apr Cmr; 8.0 kbThis study
    pHY304ori[Ts] lacZα; pBlueScript; Ermr; 4.5 kb 26
    pLM103ori[Ts] cat; 3.9-kb pDC147 ΔcpsK amplicon cloned into pHY304 EcoRV T-vector site; Ermr Cmr; 8.5 kbThis study
    pMut3 gfp(mut3) Apr 13
    pBL-gfp(mut3) gfp(mut3) Spr; 4.3 kbThis study
    pBL26ori[pAMβ1] gfp(mut3) cps-promoter; pLZ12spec derivative; Spr; 4.5 kbThis study
    pLM104 cpsK; 1.7-kb cpsK amplicon from serotype III GBS strain COH1 cloned into NcoI/HindIII-restricted pBL26; Ermr 5.6 kbThis study
    pDC132 cpsL; 1.9-kb cpsL amplicon from serotype III GBS strain COH1 cloned into pGEM T-Easy; Apr; 4.9 kbThis study
    pDC150 cpsL::BamHI; BamHI site insertion into SmaI site of pDC132; Apr; 4.9 kbThis study
    pCIV2 km-Ω-2; Kanr; 5.9 kb 32
    pDC151 cpsL::km-Ω-2; 2.2-kb pCIV2 km-Ω-2-containing amplicon cloned into BamHI-digested pDC150; Apr Kanr; 7.2 kbThis study
    pDC152 cpsL::km-Ω-2; ori[Ts]; 4.2-kb pDC151 cpsL::km-Ω-2-containing amplicon ligated to pHY304 EcoRV T-vector site; Kanr Ermr; 8.7 kbThis study
  • a Tetr, tetracycline resistance; Ermr, erythromycin resistance; Apr, ampicillin resistance; Cmr, chloramphenicol resistance; Kanr, kanamycin resistance; Spr, spectinomycin resistance; CPS, capsule negative; CPS+, capsule positive; Ts, temperature sensitive.