Queuosine (Q) is a hypermodified 7-deazaguanosine nucleoside located in the anticodon wobble position of four amino acid-specific tRNAs. In bacteria, Q is produced de novo from GTP via the 7-deazaguanosine precursor preQ1 (7-aminoethyl 7-deazaguanine) by an uncharacterized pathway. PreQ1 is subsequently transferred to its specific tRNA by a tRNA-guanine transglycosylase (TGT) and then further modified in situ to produce Q. Here we use comparative genomics to implicate four gene families (best exemplified by the B. subtilis operon ykvJKLM) as candidates in the preQ1 biosynthetic pathway. Deletions were constructed in genes for each of the four orthologs in Acinetobacter. High pressure liquid chromatography analysis showed the Q nucleoside was absent from the tRNAs of each of four deletion strains. Electrospray ionization mass spectrometry confirmed the absence of Q in each mutant strain. Finally, introduction of the Bacillus subtilis ykvJKLM operon in trans complemented the Q deficiency of the two deletion mutants that were tested. Thus, the products of these four genes (named queC, -D, -E, and -F) are essential for the Q biosynthetic pathway.