Table 1.
Strains, plasmids and oligonucleotides used in this study.
| Constructs | Genotype/description/sequence | Source |
|---|---|---|
| S. pneumoniae strains | ||
| R6 | ||
| R6 LytA-sfGFP | bgaA::PczcD –lytA (pJB3); AmpR, TetR | This work |
| R6 ΔlytA | lytA::cat; CatR | Pagliero et al. (2008) |
| ΔlytA LytA-sfGFP | lytA::cat, bgaA::PczcD –lytA (pJB3); CatR, pJB3, AmpR, TetR | This work |
| ΔlytA LytAE87Q-sfGFP | lytA::cat; bgaA::PczcD –lytAE87Q (pJB31), CatR, AmpR, TetR | This work |
| ΔlytA LytAAMI-sfGFP | lytA::cat; bgaA::PczcD –lytAAMI(pJB33); CatR, AmpR, TetR | This work |
| ΔlytA LytACBD-sfGFP | lytA::cat; bgaA::PczcD –lytACBD(pJB34); CatR, AmpR, TetR | This work |
| ftsZ-mKate | R800 ftsZ::ftsZ-mKate; StrR | Bonnet et al. (2017) |
| RH426 | ΔcomA ΔIS1167::Janus, EryRKanR | Johnsborg and Håvarstein (2009) |
| RH1 | ΔcomA::ermAM, egb::spc, EryR SpcR | Johnsborg et al. (2008) |
| RH14 | ΔcomA::ermAM, egb::spc, ΔlytA::kan, EryR SpcR KanR | Eldholm et al. (2009) |
| SPH125 | PcomR::comR, Janus between cpsO and cpsN, EryRKanR | Berg et al. (2011) |
| SPH130 | SPH125, but contains the P1 and PcomR promoters before comR | Berg et al. (2011) |
| SPH131 | SPH130, but with replacement of luc by the Janus cassette, EryRKanR | Berg et al. (2011) |
| SPH143 | SPH131 but with replacement of the Janus cassette by tarIJ (spr1149/spr1148), EryRSmR | This work |
| SPH144 | SPH143, but with replacement of native tarI and tarJ by the Janus cassette, EryRKanR | This work |
| SPH145 | SPH144, but with deletion of the Janus cassette, EryRSmR | This work |
| SPH146 | SPH145, but with replacement of lytA by a spectinomycin resistance marker (aad9), EryRSmRSpcR | This work |
| Plasmids | ||
| pADG0 | [bgaA::PZn-sfgfpopt], AmpR, TetR, AgeI, BssHII, BsiWI | Bonnet et al. (2017) |
| pJB3 | From pADG0, [bgaA::PZn-LytA-sfgfpop], AmpR, TetR | This work |
| pJB31 | From pJB3, [bgaA::PZn-LytAE87Q-sfgfpop], AmpR, TetR | This work |
| pJB33 | From pJB3, [bgaA::PZn-LytAAMI-sfgfpop], AmpR, TetR | This work |
| pJB34 | From pJB3, [bgaA::PZn-LytACBD-sfgfpop], AmpR, TetR | This work |
| pADG12 | From pET vector, 8-His-sfGFPop; Kan | This work |
| pADG14 | 8-His-sfGFPop-LytA; KanR | This work |
| pADG141 | 8-His-sfGFPop-LytAAMI; KanR | This work |
| pADG142 | 8-His-sfGFPop-LytACBD; KanR | This work |
| pET28-His-LytA | pET28a::his-lytA KanR, CmR | Philippe et al. (2015) |
| pDS-lytA | pRSET A with lytA cloned downstream of T7/lac | Berg et al. (2011) |
| Oligonucleotides | ||
| pJB3For | CGCGCGCGCATGGAAATTAATGTGAGTAAATTAAG | This work |
| pJB3Rev | GCGCGTACGTTTTACTGTAATCAAGCCATCTGG | This work |
| FORoMJ62 | ggttgaatggctttcaatcagttgaaccgctgcataggtctcagc insertion of E87Q mutation in lytA | Bonnet et al. (2017) |
| REVoMJ63 | gctgagacctatgcagcggttcaactgattgaaagccattcaacc insertion of E87Q mutation in lytA | Bonnet et al. (2017) |
| pJB33For | CAGTTTAAGCATGATATTGAGAACGGCTTGCGTACGACCGGTTCTAAAGGTGAAGAG | This work |
| pJB33Rev | CTCTTCACCTTTAGAACCGGTCGTACGCAAGCCGTTCTCAATATCATGCTTAAACTG | This work |
| pJB34For | GCGGATCTTAAGGAGGCGCGCATGACGATTGAAACAGGCTGGCAGAAG | This work |
| pJB34Rev | CTTCTGCCAGCCTGTTTCAATCGTCATGCGCGCCTCCTTAAGATCCGC | This work |
| pADG14For | CGCAAGCTT GAAATTAATGTGAGTAAATTAAGAAC | |
| pADG14REv | GCGCTCGAGTTATTATTTTACTGTAATCAAGCCATCTGG | |
| pADG141For | GCATGATATTGAGAACGGCTTGACTAATAACTC GAGCACCACCACCAC | This work |
| pADG141Rev | GTGGTGGTGGTGCTCGAGTTATTAGTCAAGCCGTTCTCAATATCATGC | This work |
| pADG142For | CCGAATTCGAGCTCCGTCGACAAGCTTGATTGAAACAGGCTGGCAGAAGAATG | This work |
| pADG142Rev | CATTCTTCTGCCAGCCTGTTTCAATCAAGCTTGTCGACGGAGCTCGAATTCGG | This work |
| khb31 | ATAACAAATCCAGTAGCTTTGG | Berg et al. (2011) |
| khb36 | TGAACCTCCAATAATAAATATAAAT | Berg et al. (2011) |
| khb33 | TTTCTAATATGTAACTCTTCCCAAT | Berg et al. (2011) |
| khb34 | CATCGGAACCTATACTCTTTTAG | Berg et al. (2011) |
| Kan484F | GTTTGATTTTTAATGGATAATGTG | Johnsborg et al. (2008) |
| LytAF | TGTATCTATCGGCAGTGTGAT | Eldholm et al. (2009) |
| LytAR | TCAACCATCCTATACAGTGAA | Eldholm et al. (2009) |
| khb90 | ATTTATATTTATTATTGGAGGTTCAATGATTTATGCAGGAATTCTTGC | This work |
| khb91 | ATTGGGAAGAGTTACATATTAGAAATTATACTTCCCACTTAAACACTG | This work |
| khb92 | AGAGACCAGCATCAGGATAC | This work |
| khb93 | CACATTATCCATTAAAAATCAAACCTATTTTCTCCTTTGTCTTAC | This work |
| khb94 | AAGTATTTTCTAGTATTATAGCACATTTAACTTTCCTTATGCTTTTGGAC | This work |
| khb95 | TTAAATGTGCTATAATACTAGAAAATACTTGTACTGGAGGTTAATTGTGGAG | This work |
| khb96 | TTCACCTTGCTCTTCCTTATAG | This work |
| khb97 | CTCCACAATTAACCTCCAGTACCTATTTTCTCCTTTGTCTTAC | This work |
| khb98 | GTAAGACAAAGGAGAAAATAGGTACTGGAGGTTAATTGTGGAG | This work |