The Escherichia coli phage 9g is resistant to many restriction enzymes and we recently found its DNA contained a novel complex modification, 2’-deoxyarchaeosine (dG+). We are now focusing on characterizing the 9g dG+ synthesis pathway. This 7-deazaguanine-derived base is normally found in tRNA of Archaea (G+). Both G+ and queuosine (Q), another 7-deazaguanine derivative found in bacterial tRNA, are synthesized from GTP through the 7-cyanodeazaguanine (preQ0) intermediate. The 9g genome encodes homologs of the preQ0 synthesis genes folE, queD and queE. These are functional when expressed in trans on multicopy plasmids and complement the dT auxotrophy and Q-phenotype of the respective E. coli mutants. A homolog of the archaeal Gat-QueC protein, previously show to be in involved in G+ synthesis, is also encoded by the 9g genome and we propose it is involved in converting preQ0 to G+ before or after its insertion in phage DNA. Attempts to validate Gat-QueC activity in a previously developed genetic test in E. coli failed so we are currently trying either to delete the gene from the phage, or to complement the G+-phenotype in the model archaeaon Haloferax volcanii. In Archaea, preQ0 is inserted into tRNA by the tRNA-guanine transglycosylase (TGT) and a TGT-like enzyme is encoded by the 9g genome. Detailed multiple alignment analysis revealed that all the phage TGT-like proteins identified contain catalytic residues and preQ0 binding residues but lack the predicted Zinc binding residues. In addition, a histidine residue is highly conserved in all these proteins, at position 196 for 9g TGT-like, but not in the bacterial or archaeal TGT. We found that the 9g TGT-like gene did not complement the Q deficiency phenotype of an E. coli tgt- strain confirming that it has functionally diverged. Current studies are in progress to identify the specific modified residue in 9g DNA using a combination of molecular, genetic and biochemical techniques.