Table S2. Bacterial strains used in this studya
Strain / Relevant genotypeb / Derivation / Selectable markerc / References/CommentsStreptococcus pneumoniae strains
EL59 / Streptococcus pneumoniae R6 parent strain / Laboratory strain derived from serotype 2 encapsulated D39 / None / A. Tomasz collection (see (1))
IU1627 / EL59 pcsB+Pc-erm / pcsB+Pc-erm x EL59 / Erm / (1)
IU1690 / Serotype 2 encapsulated D39 / NTCC7466 / None / (2)
IU1781 / IU1690 rpsL1 / rpsL1x IU1690 / Str / (3)
IU1824 / IU1690 Δcps2A’(cps2BCDETFG)H’rpsL41 / rpsL41x IU1945 / Str / (2)
IU1885 / IU1781 ΔwalK::P-kan rpsL+ / ΔwalK::P-kan rpsL+x IU1781 / Kan / (4)
IU1945 / IU1690 Δcps2A’(cps2BCDETFG)H’ / Δcps2A’(cps2BCDETFG)H’ x IU1690 / None / (2)
IU3176 / IU1781 ΔwalJ / ΔwalJx IU1885 / Str / (4)
IU3900 / IU1945 lytA>Pc-aad9 / lytA>Pc-aad9x IU1945 / Spec / (5)
IU4081 / IU1945 pcsBW335GPc-erm / pcsBW335G Pc-ermx IU1945 / Erm / (6)
IU4240 / IU1781 ΔwalJCEP::P-kan rpsL+ / CEP::P-kan rpsL+x IU3176 / Kan / (4)
IU4256 / IU1945 pcsBA160PPc-erm / pcsBA160PPc-ermx IU1945 / Erm / (6)
IU4261 / IU1945 pcsBL78S L219P Pc-erm / pcsBL78S L219P Pc-ermx IU1945 / Erm / (6)
IU4262 / IU1945 pcsBA160PPc-erm / pcsBA160PPc-ermx IU1945 / Erm / (6)
IU4321 / IU1781 ΔwalJ CEP::PfcsK-walJ / CEP::PfcsK-walJ x IU4240 / Str / (4)
IU4325 / IU1945 ftsEX+ P-kan rpsL+ / ftsEX+ P-kan rpsL+ x IU1945 / Kan / This study
IU5122 / IU1824 CEP::P-kan rpsL+ / CEP::P-kan rpsL+x IU1824 / Kan / This study
IU5382 / IU1824 CEP::PfcsK-ftsE+ (walJ terminator) / CEP::PfcsK-ftsE+x IU5122 / Str / This study
IU5677 / IU1945 ftsX Y254C I300T P-kan rpsL+ / ftsEXM P-kanrpsL+d x IU1945 / Kan / ftsEX(Ts) mutant
IU5678 / IU4261ftsXQ268E P-kan rpsL+ / ftsEXM P-kanrpsL+x IU4261 / Kan, Erm / Isolated suppressor of Ts pcsB L78S L219P (IU4261)
IU5679 / IU4261ftsXA177V P-kan rpsL+ / ftsEXM P-kanrpsL+x IU4261 / Kan, Erm / Isolated suppressor of Ts pcsB L78S L219P (IU4261)
IU5690 / IU4261 ftsXQ268EP-kan rpsL+ / ftsXQ268E P-kan rpsL+ x IU4261 / Kan, Erm / Backcross of suppressor of Ts pcsB L78S L219P (IU5678)
IU5692 / IU4261ftsXA177VP-kan rpsL+ / ftsXA177V P-kan rpsL+x IU4261 / Kan, Erm / Backcross of suppressor of Ts pcsB L78S L219P (IU5679)
IU5694 / IU1945 ftsX Y254C I300TP-kan rpsL+ / ftsX Y254C I300TP-kan rpsL+x IU1945 / Kan / Backcross of ftsEX(Ts) mutant IU5677
IU5756 / IU1824 ΔftsE::P-erm // CEP::PfcsK-ftsE+ / ΔftsE::P-erme x IU5382 / Erm / This study
IU5818 / IU1824 ΔftsE::P-kan rpsL+ // CEP::PfcsK-ftsE+ / ΔftsE::P-kan rpsL+ x IU5382 / Kan / This study
IU5910 / IU4256 ftsXF179S P-kan rpsL+ / ftsEXM P-kanrpsL+ x IU4256 / Kan, Erm / Isolated suppressor of Ts pcsBA160P (IU4256)
IU5961 / IU4256ftsXF179S P-kan rpsL+ / ftsXF179S P-kan rpsL+x IU4256 / Kan, Erm / Backcross of suppressor of Ts pcsBA160P (IU5910)
IU6001 / IU1945 ftsXI150K P-kan rpsL+ / ftsEXM P-kanrpsL+ x IU1945 / Kan / ftsEX(Ts) mutant
IU6023 / IU1824 ftsEK43Q//CEP::PfcsK-ftsE+ / ftsEK43Q x IU5818 / Str / This study
IU6025 / IU1824 ftsED164A//CEP::PfcsK-ftsE+ / ftsED164Ax IU5818 / Str / This study
IU6027 / IU1824 ftsEE165A//CEP::PfcsK-ftsE+ / ftsEE165Ax IU5818 / Str / This study
IU6039 / IU1945 ftsXI150K P-kan rpsL+ / ftsXI150K P-kan rpsL+ x IU1945 / Kan / Backcross of ftsEX(Ts) mutant IU6001
IU6157 / IU1945 ftsXI150K P-kan rpsL+ / ftsEXM P-kanrpsL+ x IU1945 / Kan / ftsEX(Ts) mutant
IU6159 / IU1945 ftsXI150K P-kan rpsL+ / ftsEXM P-kanrpsL+ x IU1945 / Kan / ftsEX(Ts) mutant
IU6161 / IU1945 ftsXI150K P-kan rpsL+ / ftsEXM P-kanrpsL+ x IU1945 / Kan / ftsEX(Ts) mutant
IU6163 / IU1945 ftsXI150K P-kan rpsL+ / ftsEXM P-kanrpsL+ x IU1945 / Kan / ftsEX(Ts) mutant
IU6165 / IU1945 ftsXI150K P-kan rpsL+ / ftsEXM P-kanrpsL+ x IU1945 / Kan / ftsEX(Ts) mutant
IU6167 / IU1945 ftsXI150K P-kan rpsL+ / ftsEXM P-kanrpsL+ x IU1945 / Kan / ftsEX(Ts) mutant
IU6169 / IU1945 ftsXI150K P-kan rpsL+ / ftsEXM P-kanrpsL+ x IU1945 / Kan / ftsEX(Ts) mutant
IU6171 / IU1945 ftsXI150K P-kan rpsL+ / ftsEXM P-kanrpsL+ x IU1945 / Kan / ftsEX(Ts) mutant
IU6179 / IU1945 ftsXI150K P-kan rpsL+ / ftsEXM P-kanrpsL+ x IU1945 / Kan / Backcross of ftsEX(Ts) mutant IU6163
IU6254 / IU4256 ftsXS264LP-kan rpsL+ / ftsEXM P-kanrpsL+ x IU4256 / Kan, Erm / Isolated suppressor of Ts pcsBA160P (IU4256)(also suppresses TspcsBL78S L219P)
IU6270 / IU4256 ftsXS264L P-kan rpsL+ / ftsXS264LP-kan rpsL+xIU4256 / Kan, Erm / Backcross of suppressor of Ts pcsBA160P (IU6254)
IU6321 / IU4256 ftsXW183LP-kan rpsL+ / ftsEXMP-kan rpsL+ x IU4256 / Kan, Erm / Isolated suppressor of Ts pcsBA160P (IU4256) (also suppresses Ts pcsBL78S L219P)
IU6333 / IU4256 ftsXW183L P-kan rpsL+ / ftsXW183L P-kan rpsL+x IU4256 / Kan, Erm / Backcross of suppressor of Ts pcsBA160P in ftsEX(IU6321)
IU6335 / IU4261 ftsXW183L P-kan rpsL+ / ftsXW183L P-kan rpsL+x IU4261 / Kan, Erm / Allele from IU6321, backcross of suppressor of Ts pcsB L78S L219P (IU6349)
IU6337 / IU1945 ftsXI150K P-kan rpsL+
backcrossed / ftsEXM P-kanrpsL x IU1945 / Kan / Backcross of ftsEX(Ts) mutant IU6159
IU6338 / IU1945 ftsXI150K P-kan rpsL+
backcrossed / ftsEXM P-kanrpsL x IU1945 / Kan / Backcross of ftsEX(Ts) mutant IU6161
IU6339 / IU1945 ftsXI150K P-kan rpsL+
backcrossed / ftsEXM P-kanrpsL x IU1945 / Kan / Backcross of ftsEX(Ts) mutant IU6165
IU6340 / IU1945 ftsXI150K P-kan rpsL+
backcrossed / ftsEXM P-kanrpsLx IU1945 / Kan / Backcross of ftsEX(Ts) mutant IU6167
IU6341 / IU1945 ftsXI150K P-kan rpsL+
backcrossed / ftsEXM P-kanrpsL x IU1945 / Kan / Backcross of ftsEX(Ts) mutant IU6169
IU6342 / IU1945 ftsXI150K P-kan rpsL+
backcrossed / ftsEXM P-kanrpsL x IU1945 / Kan / Backcross of ftsEX(Ts) mutant IU6171
IU6345 / IU4261 ftsXS264L P-kan rpsL+ / ftsEXM P-kanrpsL+ x IU4261 / Kan, Erm / Isolated suppressor of Ts pcsB L78S L219P (IU4261)
IU6346 / IU4261 ftsXV266A P-kan rpsL+ / ftsEXM P-kanrpsL+ x IU4261 / Kan, Erm / Isolated suppressor of Ts pcsB L78S L219P (IU4261)
IU6347 / IU4261 ftsXF179S P-kan rpsL+ / ftsEXM P-kanrpsL+ x IU4261 / Kan, Erm / Isolated suppressor of Ts pcsB L78S L219P (IU4261)
IU6348 / IU4261 ftsXK88M R204H P-kan rpsL+ / ftsEXM P-kanrpsL+ x IU4261 / Kan, Erm / Isolated suppressor of Ts pcsB L78S L219P (IU4261)
IU6349 / IU4261 ftsXW183L P-kan rpsL+ / ftsEXM P-kanrpsL+ x IU4261 / Kan, Erm / Isolated suppressor of Ts pcsB L78S L219P (IU4261)
IU6351 / IU4261 ftsXA177V I192V D276Q P-kan rpsL+ / ftsEXM P-kanrpsL+ x IU4261 / Kan, Erm / Isolated suppressor of Ts pcsB L78S L219P (IU4261)
IU6360 / IU1945 ftsXI150K P-kan rpsL+ / ftsXI150KP-kan rpsL+x IU1945 / Kan / Backcross of ftsEX(Ts) mutant IU6157
IU6361 / IU4261 ftsXS264L P-kan rpsL+ / ftsXS264L P-kan rpsL+ x IU4261 / Kan, Erm / Backcross of suppressor of Ts pcsB L78S L219P (IU6345)
IU6363 / IU4261 ftsXV266A P-kan rpsL+ / ftsXV266A P-kan rpsL+x IU4261 / Kan, Erm / Backcross of suppressor of Ts pcsB L78S L219P (IU6346)
IU6365 / IU4261 ftsXF179S P-kan rpsL+ / ftsXF179S P-kan rpsL+x IU4261 / Kan, Erm / Backcross of suppressor of Ts pcsB L78S L219P (IU6347)
IU6367 / IU4261 ftsXK88M R204H P-kan rpsL+ / ftsXK88M R204H P-kanx IU4261 / Kan, Erm / Backcross of suppressor of Ts pcsB L78S L219P (IU6348)
IU6371 / IU4261 ftsXS264L P-kan rpsL+ / ftsXS264L P-kan rpsL+ x IU4261 / Kan, Erm / This study
IU6385 / IU4261 ftsXA177V I192V D276Q P-kan rpsL+ / ftsXA177V I192V D276Q P-kan rpsL+ x IU4261 / Kan, Erm / Backcross of suppressor of Ts pcsB L78S L219P (IU6351)
IU6387 / IU4256 ftsXA177V I192V D276Q P-kan rpsL+ / ftsXA177V I192V D276Q P-kan rpsL+ x IU4256 / Kan, Erm / This study
IU6426 / IU1945 ftsXS264L P-kan rpsL+ / ftsXS264LP-kan rpsL+x IU1945 / Kan / This study
IU6427 / IU1945 ftsXF179SP-kan rpsL+ / ftsXF179SP-kan rpsL+x IU1945 / Kan / This study
IU6429 / IU1945 ftsXW183LP-kan rpsL+ / ftsXW183LP-kan rpsL+x IU1945 / Kan / This study
IU6434 / IU1945 ftsXQ268E P-kan rpsL+ / ftsXQ268EP-kan rpsL+x IU1945 / Kan / This study
IU6437 / IU1945 ftsXA177V P-kan rpsL+ / ftsXA177V P-kan rpsL+x IU1945 / Kan / This study
IU6439 / IU1945 ftsXV266A P-kan rpsL+ / ftsXV266AP-kan rpsL+ x IU1945 / Kan / This study
IU6500 / IU4262 ftsXA177V P-kan rpsL+ / ftsXA177V P-kan rpsL+x IU4262 / Kan, Erm / Isolated suppressor of Ts pcsBA160P (IU4262)
IU6502 / IU4262 ftsXW183LP-kan rpsL+ / ftsXW183LP-kan rpsL+x IU4262 / Kan, Erm / This study
IU6525 / IU4262 ftsXA177V P-kan rpsL+ / ftsXA177VP-kan rpsL+x IU4262 / Kan, Erm / Backcross of suppressor of Ts pcsBA160P (IU6500)
E014 / IU1945 Δspr_0286::P-erm / Δspr_0286::P-erm x IU1945 / Erm / (6)
K003 / IU1945 ΔcbpF::P-kan rpsL+ / ΔcbpF::P-kan rpsL+x IU1945 / Kan / (6)
K043 / IU1945 ΔlytA::P-kan rpsL+ / ΔlytA::P-kan rpsL+x IU1945 / Kan / (5)
aStrains were constructed by transformation of amplicons (left of “×”) into the indicated recipient strain (right of “×”) as described in Materials and Methods. > or :: indicates exact replacement of a reading frame or insertion, respectively. Primers for constructing the strains are listed in Table S3.
b“Pc” refers to the constitutive synthetic promoter that drives the erythromycin resistant gene (8). “PfcsK” refers to the fucose operon promoter, and “P” is a constitutive promoter in the Janus cassette (see (7)).
cAntibiotic resistance markers: Erm, erythromycin; Kan, kanamycin; Spec, spectinomycin; Str: streptomycin; Cm: chloramphenicol.
dftsEXMP-kan rpsL+amplicon contained 0-4 mutations per kb generated by error-prone PCR (see Materials and Methods).
eIn ftsE or ∆ftsX mutations, 60 bp of the ftsE or ftsX reading frame was left intact on the 5’ and 3’ junctions surrounding the inserted antibiotic resistance cassettes to maintain adjacent genetic signals.
References
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