The Widely Conserved ebo Cluster Is Involved in Precursor Transport to the Periplasm during Scytonemin Synthesis in Nostoc punctiforme

Scytonemin is a dimeric indole-phenol sunscreen synthesized by some cyanobacteria under conditions of exposure to UVA radiation. While its biosynthetic pathway has been elucidated only partially, comparative genomics reveals that the scytonemin operon often contains a cluster of five highly conserved genes (ebo cluster) of unknown function that is widespread and conserved among several bacterial and algal phyla. We sought to elucidate the function of the ebo cluster in the cyanobacterium Nostoc punctiforme by constructing and analyzing in-frame deletion mutants (one for each ebo gene and one for the entire cluster). Under conditions of UVA induction, all ebo mutants were scytoneminless, and all accumulated a single compound, the scytonemin monomer, clearly implicating all ebo genes in scytonemin production. We showed that the scytonemin monomer also accumulated in an induced deletion mutant of scyE, a non-ebo scytonemin gene whose product is demonstrably targeted to the periplasm. Confocal autofluorescence microscopy revealed that the accumulation was confined to the cytoplasm in all ebo mutants but that that was not the case in the scyE deletion, with an intact ebo cluster, where the scytonemin monomer was also excreted to the periplasm. The results implicate the ebo cluster in the export of the scytonemin monomer to the periplasm for final oxidative dimerization by ScyE. By extension, the ebo gene cluster may play similar roles in metabolite translocation across many bacterial phyla. We discuss potential mechanisms for such a role on the basis of structural and phylogenetic considerations of the ebo proteins.