Epigenetic control of tetrapyrrole biosynthesis by m4C DNA methylation in a cyanobacterium

Epigenetic DNA modifications are pivotal in eukaryotic gene expression, but their regulatory significance in bacteria is less understood. In Synechocystis 6803, the DNA methyltransferase M.Ssp6803II modifies the first cytosine in the GGCC motif, forming N4-methylcytosine (GGm4CC). Deleting the sll0729 gene ({Delta}sll0729) caused a bluish phenotype due to reduced chlorophyll levels, which was reversed by suppressor mutations. Re-sequencing of seven suppressor clones revealed a common GGCC to GGTC mutation in the slr1790 promoters discriminator sequence, encoding protoporphyrinogen IX oxidase, HemJ, crucial for tetrapyrrole biosynthesis. Transcriptomic and qPCR analyses indicated aberrant slr1790 expression in {Delta}sll0729 mutants. This aberration led to the accumulation of coproporphyrin III and protoporphyrin IX, indicative of impaired HemJ activity. To confirm the importance of DNA methylation in hemJ expression, native and mutated hemJ promoter variants were introduced into the wild type, followed by sll0729 deletion. The sll0729 deletion segregated in strains with the GGTC motif in the hemJ promoter, resulting in wild-type-like pigmentation, whereas freshly prepared {Delta}sll0729 mutants with the native hemJ promoter exhibited the bluish phenotype. These findings demonstrate that hemJ is tightly regulated in Synechocystis and that N4-methylcytosine is essential for proper hemJ expression.