Figure 2.—

Complementation of the ccs4 mutant. (A, B, and D) The ccs4-F2D8 arg7-8 strain was transformed with pSL18 (ccs4), pSL18 carrying a 1-kb genomic fragment with the CCS4 gene (CCS4), and pSL18 expressing the full-length CCS4 coding sequence (CCS4, ORF1) or a truncated form of the CCS4 protein (CCS4, ORF2). (C) The ccs4-F2D8 arg7-8 strain was transformed with pCB412 (ccs4) or cotransformed with pCB412 and pSK-CCS4-SacII carrying a 1-kb genomic fragment with the CCS4 gene [ccs4 (CCS4)]. Only one representative transformant is shown in A–C. In D, two representative transformants (CCS4, ORF2) are shown. In B–D, CC124 is the wild-type strain (WT). (A) Restoration of the photosynthetic growth of ccs4 by full-length and truncated CCS4. Ten-fold dilution series of each transformant were plated on acetate (under heterotrophic conditions, 20 μmol/m²/sec of light) and minimal medium (phototrophic conditions, 250 μmol/m²/sec of light) and incubated at 25° for 1 and 3 weeks, respectively. (B) Fluorescence kinetics indicate restoration of cytochrome b₆f in ccs4 complemented with the full-length and truncated CCS4 gene. Fluorescence transients were measured on colonies grown for 1 day on solid acetate medium after a short dark adaptation using Handy Fluorcam (Photon System Instruments). The fluorescence is in arbitrary units (A.U.) and recorded over a 3-sec illumination period. (C) Plastid c-type cytochromes accumulation is restored in ccs4 complemented with the CCS4 gene.Strains were analyzed for cytochrome f and cytochrome c₆ accumulation by heme stain and immunoblot. Samples corresponding to 18 μg of chlorophyll were separated in 12% SDS acrylamide gel to detect cytochrome f and CF₁ that serves as loading control. Samples corresponding to 16 μg of chlorophyll were separated in 15% native acrylamide gel to detect cytochrome c₆. For an estimation of the protein abundance in the ccs4-complemented strain, dilutions of the wild-type sample were loaded on the gel. Gels were transferred to PVDF membranes prior to heme staining and immunodetection with antisera against cytochrome f, cytochrome c₆, and CF₁. (D) Cytochrome f accumulation is partially restored in ccs4 complemented by a truncated form of the CCS4 gene. Strains were analyzed for cytochrome f accumulation via heme stain and immunoblot. Experimental conditions are the same as described in C.