Identification of the minimal bacterial 2'-deoxy-7-amido-7-deazaguanine synthesis machinery

The 7-deazapurine derivatives, 2'-deoxy-7-cyano-7-deazaguanosine (dPreQ₀ ) and 2'-deoxy-7-amido-7-deazaguanosine (dADG) are recently discovered DNA modifications encoded by the dpd cluster found in a diverse set of bacteria. Here we identify the genes required for the formation of dPreQ₀ and dADG in DNA and propose a biosynthetic pathway. The preQ₀ base is a precursor that in Salmonella Montevideo, is synthesized as an intermediate in the pathway of the tRNA modification queuosine. Of the 11 genes (dpdA - dpdK) found in the S. Montevideo dpd cluster, dpdA and dpdB are necessary and sufficient to synthesize dPreQ₀ , while dpdC is additionally required for dADG synthesis. Among the rest of the dpd genes, dpdE, dpdG, dpdI, dpdK, dpdD and possibly dpdJ appear to be involved in a restriction-like phenotype. Indirect competition for preQ₀ base led to a model for dADG synthesis in which DpdA inserts preQ₀ into DNA with the help of DpdB, and then DpdC hydrolyzes dPreQ₀ to dADG. The role of DpdB is not entirely clear as it is dispensable in other dpd clusters. Our discovery of a minimal gene set for introducing 7-deazapurine derivatives in DNA provides new tools for biotechnology applications and demonstrates the interplay between the DNA and RNA modification machineries.