TABLE 1.
Strains and Plasmids used in this work
| Strains/Plasmids | Relevant characteristicsa | References |
|---|---|---|
| Bacteria | ||
| R. capsulatus | ||
| MT1131 | Wild type, ctrD121 Rifr Res+ Nadi+ Ps+ | (58) |
| MT-SRP1 | Δ (ccmI::kan) Res+ Nadi− Ps− | (11) |
| Escherichia coli | ||
| HB101 | F− Δ(gpt-proA)62 araC14 leuB6(Am) glnV44(AS) galK2(Oc) lacY1 Δ(mcrC-mrr) rpsL20(Strr) xylA5 mtl-1 thi-1 | Stratagene |
| XL1-Blue | endA1 gyrA96(NalR) thi-1 recA1 relA1 lac glnV44 F′[::Tn10 proAB+lacIq Δ(lacZ)M15] hsdR17(rK−mK+) | Stratagene |
| RP4182 | trp, gal, rpsL Δ(supE, dcm, fla) | (59) |
| Plasmids | ||
| pBSK | pBluescriptIISK+, Ampr | Stratagene |
| pRK415 | Broad host-range vector, gene expression mediated by E. coli lacZ promoter, Tetr | (60) |
| pCHB500 | Broad host-range vector with R. capsulatus cycA promoter, Tetr | (61) |
| pRK404 | Broad host-range vector, Tetr | (60) |
| pCS1302 | pCS905 derivative, Strep-tag II sequence fused to GFP, rendering GFP replaceable by cloning any gene of interest in-frame into NdeI and BamHI sites, Ampr | (7) |
| pCS905 | pET-3a derivative (Novagen) with T7 promoter region replaced by a DNA fragment encoding lacI and the tac promoter region, Ampr | |
| pCS1726 | R. capsulatus cycA encoding mature cyt c2 with a N-terminal Strep-tag cloned into pCS1302 using NdeI and BamHI sites, Ampr | (27) |
| pSVEN | 1.65-kb StuI-BbsI fragment containing R. capsulatus CcmI cloned in pRK404 | (11) |
| pAV1 | pCS1726 derivative with an additional in-frame stop codon (TAA) 15 bp downstream from the native TAG of cycA, Ampr | This work |
| pAV1C13S | Cys-13 of R. capsulatus cycA in pAV1 mutated to Ser, Ampr | This work |
| pAV1C16S | Cys-16 of R. capsulatus cycA in pAV1 mutated to Ser, Ampr | This work |
| pAV1H17S | His-17 of R. capsulatus cycA in pAV1 mutated to Ser, Ampr | This work |
| pAV1M96S | Met-96 of R. capsulatus cycA in pAV1 mutated to Ser, Ampr | This work |
| pAV1C13SC16S | Cys-13 and Cys-16 of R. capsulatus cycA in pAV1 mutated to Ser, Ampr | This work |
| pAV2 | pAV1 derivative with an in-frame stop codon (TAA) 78-bp upstream of the native TAG codon, deleting the 26 last amino acids of cycA, Ampr | This work |
| pAV2C13SC16S | Cys-13 and Cys-16 of R. capsulatus cycA in pAV2 mutated to Ser, Ampr | This work |
| pAV3 | pAV1 derivative with an in-frame stop codon (TAA) 42 bp upstream of the native TAG codon, deleting the 15 last amino acids of cycA, Ampr | This work |
| pCS1303 | pCS905 derivative, His10-tag sequence fused to GFP, rendering GFP replaceable by cloning any gene of interest in-frame into NdeI and BamHI sites, Ampr | This work |
| pMADO3 | PCR amplified full length ccmI with introduced 5′-NdeI and 3′-BamHI sites, phosphorylated and cloned into EcoRV-restricted pBSK, Ampr | This work |
| pMADO5 | ccmI from pMADO3 cloned into NdeI-BamHI in pCS1303, Ampr | This work |
| pMADO5–1 | pCS1303 derivative with a 360-bp 5′-NdeI and 3′-BamHI PCR fragment of R. capsulatus MT1131 genomic DNA, yielding a truncated ccmI with its first 121 amino acid residues (CcmI-1 domain), Ampr | This work |
| pMADO5–2 | pMADO5 derivative, obtained by in-frame deletion of the first 250 bp, yielding a ccmI derivative lacking the first 83 amino acid residues (CcmI-2 domain), Ampr | This work |
| pCS1587 | 1.36-kb XbaI-BamHI fragment from pMADO5 cloned into pCHB500 | This work |
a Res and Ps refer to respiratory and photosynthetic growth, respectively. Nadi stain, α-naphthol + N,N-dimethyl-p-phenylenediamine, yielding indophenol blue in the presence of O2 to reveal cytochrome c oxidase activities of bacterial colonies. R. capsulatus MT1131 strain is referred to as a wild-type strain with respect to its cytochrome c profile and growth properties.