Figure 3.

Generation of synToc75 (slr1227) mutants. (A) The wild-type gene synToc75 (slr1227) of Synechocystis sp. PCC 6803 was disrupted either by an insertion of the kanamycin-resistance gene, kan^r, into synToc75 (synToc75-kan^r) or by a replacement of the synToc75 ORF by the kan^r ORF (ΔsynToc75-kan^rORF). Only the data obtained for the sense orientation of the kan^r insertion are shown (synToc75-kan^r). (B) Segregation of wild-type synToc75 was analyzed by DNA gel blotting. Transformed cells were grown under photoautotrophic conditions and transferred to new plates for the number of passages indicated. (C) As a control for genome segregation, wild-type cells were transformed with a plasmid copy of psaC, containing an insertion of the kan^r gene, increasing the size of the EcoRI fragment from 8.0 kb (wild type, wt) to 9.4 kb. Transformants were grown under photoautotrophic or light-activated heterotrophic conditions; data of the sixth and the third passage, respectively, are presented. (D) In the presence of pRL1342-encoded SynToc75 and selection with erythro- mycin and kanamycin, the chromosomal wild-type copies of synToc75 disappeared, as indicated by the absence of the 4.6-kb EcoRI fragment (synToc75-kan^r) and also the 10.5-kb fragment (ΔsynToc75-kan^rORF). The control (synToc75-kan^r) shows the presence of chromosomal wild-type and mutant synToc75. Only data for photoautotrophically grown Synechocystis transformants are presented.