Genotyping and phenotyping of Synechocystis 6803 strains to verify the importance of GGCC methylation for hemJ expression. (A) Genotyping via PCR. Upper panel, the gene encoding M.Ssp6803II is not present in strains with ∆sll0729 background as indicated by the lack of the 827 bp amplicon (generated by primers P22/P23). Lower panel, presence of the wild-type (WT) or the mutated hemJ promoter in the generated strains as indicated by the 639 bp amplicon in WT and ∆sll0729, or the 2,687 bp fragment in the manipulated strains (primers P15/P21). The latter fragment is larger due to the aadA antibiotic resistance cassette inserted 199 nt upstream of the transcription start site. Three independent clones were analyzed per strain. For details of the respective strains, see Supplementary Table S2. A schematic representation of the mutation strategy and the primer-binding sites can be found in the Supplementary Fig. S9. (B) Sequence analysis to verify the intact GGCC motif in the native promoter and its change to GGTC in the clones with mutated promoter sequence. (C) Absorption spectra of WT and ∆sll0729 grown under standard conditions were measured with a SPECORD^® 210 PLUS (Analytik Jena) spectrophotometer. (D) Absorption spectra of the indicated strains grown under standard conditions, measured using a Cary 50 Bio UV-visible (Varian) spectrophotometer. In both panels C,D the optical appearance of the corresponding cultures is shown by the circular insets. The spectra were normalized to the absorption at 750 nm. For the optical appearance of the different strains, see Supplementary Fig. S4, and for the growth of the different strains, see Supplementary Fig. S5.