Table 1.
Strains and plasmids used in this work
| Strains/plasmids | Relevant propertiesa | Refs. |
|---|---|---|
| Bacteria | ||
| R. capsulatus | ||
| MT-SRP1.r1 | Δ(ccmI::kan) G488A up-promoter of ccmFH; Res+ Ps+ | 50 |
| MD11 | Δ(ccmG::kan); Res+ Ps− | 51 |
| MD14 | ccmH::spe; Res+ Ps− | 50 |
| E. coli | ||
| HB101 | F− Δ (gpt-proA)62 araC14 leuB6(Am) glnV44(AS) thi-1 galK2(Oc) lacY1 Δ(mcrC-mrr) rpsL20(Strr) xylA5 mtl-1 | Stratagene |
| XL1 Blue | endA1 gyrA96(NalR) thi-1 recA1 relA1 lac glnV44 F′[::Tn10 proAB+ lacIq Δ(lacZ)M15] hsdR17(rK−mK+) | Stratagene |
| BL21(DE3) | fhuA2 [lon] ompT gal (λ DE3) [dcm] ΔhsdS λ DE3 = λ sBamHI oΔEcoRI-B int:: (lacI::PlacUV5::T7 gene1) i21 Δnin5 | New England Biolabs |
| Plasmids | ||
| pAV1 | pCS1302 derivative carrying truncated R. capsulatus cycA at the NdeI–BamHI sites with a Strep-tag II at its 5′-end and without a signal sequence, AmpR (Strep-apocyt c2WT)b | 29 |
| pAV10 | pFLAG-1 derivative carrying truncated R. capsulatus ccmH lacking its signal sequence and TM helix (C-terminal 46 amino acids) from pCS1604 at the NdeI–BamHI sites, AmpR (FLAG-CcmHWT) | This work |
| pAV10C42S | pAV10 derivative with C42S mutation in R. capsulatus ccmH, AmpR (FLAG-CcmHCys-45) | This work |
| AV10C45S | pAV10 derivative with C45S mutation in R. capsulatus ccmH, AmpR (FLAG-CcmHCys-42) | This work |
| pAV10C42SC45S | pAV10 derivative with C42S and C45S mutations in R. capsulatus ccmH, AmpR (FLAG-CcmH*) | This work |
| pBSK | AmpR | Stratagene |
| pCS1552 | pQE60-helX derivative with C75S mutation in R. capsulatus ccmG, AmpR | This work |
| pCS1553 | pQE60-helX derivative with C78S mutation in R. capsulatus ccmG, AmpR | This work |
| pCS1554 | pQE60-helX derivative with C75S and C78S mutations in R. capsulatus ccmG, AmpR | This work |
| pCS1555 | A truncated R. capsulatus ccmG from pQE60-helX cloned into the EcoRI–HindIII sites of pBSK, AmpR (His6–CcmGWT) | This work |
| pCS1556 | A truncated R. capsulatus ccmG with C75S mutation taken from pCS1552 and cloned into the EcoRI–HindIII sites of pBSK, AmpR (His6-CcmGCys-78) | This work |
| pCS1557 | A truncated R. capsulatus ccmG with C78S mutation taken from pCS1553 and cloned into the EcoRI–HindIII sites of pBSK, AmpR (His6-CcmGCys-75) | This work |
| pCS1558 | A truncated R. capsulatus ccmG with C75S and C78S mutations, taken from pCS1554 and cloned into the EcoRI–HindIII sites of pBSK, AmpR (His6-CcmG*) | This work |
| pCS1566 | pCHB500 derivative producing R. capsulatus ccmG with its signal peptide and His6-tagged at its 3′-end, cloned into the XbaI/KpnI sites, TetR (His6-CcmGWT) | 51 |
| pCS1604 | pACCyc3 derivative (52), producing a truncated R. capsulatus ccmH from pRGK291 (13), lacking both its signal sequence and TM helix (last 46 amino acids at its 3′-end), cloned into NdeI-BamHI sites. ClmR | This work |
| pFLAG-1 | pMADO5 derivative carrying 5′-FLAG-tag ccmI (instead of His10 tag), AmpR | 31 |
| pMADO5 | pCS1303 derivative, containing R. capsulatus ccmI carrying 5′-His10 tag, AmpR (His10-CcmIWT) | 29 |
| pMAM1 | pCS1302 derivative, containing truncated R. capsulatus petC* (derivative of petC with no signal sequence and lacking its last C-terminal 39 amino acids, and its non-heme ligating Cys-144 and Cys-167 residues) cloned into the NdeI–BamHI sites with a Strep-tag II fused at its 5′-end, AmpR (Strep-apocyt c1WT) | 30 |
| pMAM1C34S | pMAM1 derivative with C34S mutation of R. capsulatus petC*, AmpR (Strep-apocyt c1Cys-37) | This work |
| pMAM1C37S | pMAM1 derivative with C37S mutation of R. capsulatus petC*, AmpR (Strep-apocyt c1Cys-34) | This work |
| pMAM1C34SC37S | pMAM1 derivative with C34S and C37S mutations of R. capsulatus petC*, AmpR (Strep-apocyt c1*) | 30 |
| pNJ2 | pRK415 derivative producing R. capsulatus 5′-end FLAG-tagged ccmI with its own promoter, TetR (FLAG-CcmIWT) | 51 |
| pQE60 | Ampr | Qiagen |
| pQE60-helX | pQE60 derivative containing a truncated R. capsulatus ccmG without its signal sequence and with a C-terminal His6 tag cloned into the NcoI–BamHI sites | c |
| pST6 | pCHB500 derivative producing R. capsulatus ccmH, with a Strep-tag fused at its 3′-end, TetR (Strep-CcmHWT) | 14 |
a Res and Ps refer to respiratory and photosynthetic growth, respectively.
b Proteins produced by appropriate plasmids are indicated in bold fonts.
c F. Daldal, unpublished data.