aroF and cm2: potential molecular markers for the detection of stone-inhabiting Actinobacteria on cultural heritage sites

Tangible archeological sites and stone monuments are naturally decayed and deteriorated over time, providing substances that can sustain life, although they provide a complicated ecosystem characterized by low nutrition and desiccation. Stone-inhabiting bacteria (SIB) and especially members of the phylum Actinobacteria dominate such environments, particularly the members of the family Geodermatophilaceae. We used the published data of two confirmed SIB species to mine their genomes for specific molecular markers to rapidly survey the presence of SIB in cultural heritage material prior to further analysis. The search focused on the mycosporine-like amino acids (MAAs) synthesis pathway. MAAs are intracellular compounds biosynthesized by the shikimic acid pathway to synthesize aromatic amino acids and were found related to abiotic resistance features in microorganisms. Based on genome mining, the DAHP II (aroF) and a homolog of the Chorismate mutase gene (cm2) were found mostly in Actinobacteria and few other species. After calibration on five stone-inhabiting Actinobacteria (SIAb) species using conventional PCR, newly designed primers were successfully applied to environmental DNA extracted from two Egyptian pyramidal sites using a qPCR approach. This is the first report of aroF and cm2 as qPCR markers to detect SIAb from cultural heritage material prior to proceeding with further analysis (e.g., metagenomics and meta-barcoding analyses).