Metagenome-resolved functional traits of Rubrobacter species implicated in rosy discoloration of ancient frescoes in two Georgian Cathedrals

Pink biofilm formation on stone monuments and mural paintings poses serious harm to cultural heritage preservation. Pink biofilms are globally widespread and recalcitrant to eradication, often causing recurrences after restoration. Yet, the ecological drivers of pink biofilm formation and the metabolic functions sustaining the growth of pigment-producing biodeteriogens remain unclear. In this study, a combined approach integrating physicochemical investigations, scanning electron microscopy, 16S rRNA sequence-based analysis of the prokaryotic community, metagenomic deep sequencing, and metabolic profiling, was applied to determine the etiology of rosy discoloration of ancient frescoes in the Gelati and the Martvili Cathedrals (Georgia). Martvili samples showed greater diversity than Gelati samples, though Actinomycetota predominated in both samples. Rubrobacter-related sequences were detected in all sampling sites, showing an overwhelming abundance in Gelati samples. Reconstruction of metagenome-assembled genomes (MAGs) and phylogenetic analyses highlighted significant intra-genus diversity for Rubrobacter-related sequences, most of which could not be assigned to any formally described Rubrobacter species. Metabolic profiling of the Gelati metagenomes suggests that carbon-fixing autotrophic bacteria and proteinaceous substances in the plaster could contribute to sustaining the chemoorganotrophic members of the community. Complete pathways for β-carotene and bacterioruberin synthesis were identified in Rubrobacter MAGs, consistent with the Raman spectroscopy-based detection of these pigments in fresco samples. Gene clusters for the synthesis of secondary metabolites endowed with antibiotic activity were predicted from the annotation of Rubrobacter MAGs, along with genes conferring resistance to several antimicrobials and biocides. In conclusion, genome-resolved metagenomics provided robust evidence of a causal relationship between contamination by Rubrobacter-related carotenoid-producing bacteria and the rosy discoloration of Georgian frescoes, with relevant implications for rational biodeteriogen-targeted restoration strategies.