Queuosine (Q) stands out as the sole tRNA modification that can be synthesized via salvage pathways. Comparative genomic analyses identified specific bacteria that showed a discrepancy between the projected Q salvage route and the predicted substrate specificities of the two identified salvage proteins: (1) the distinctive enzyme tRNA guanine-34 transglycosylase (bacterial TGT, or bTGT), responsible for inserting precursor bases into target tRNAs; and (2) queuosine precursor transporter (QPTR), a transporter protein that imports Q precursors. Organisms such as the facultative intracellular pathogen Bartonella henselae , which possess only bTGT and QPTR but lack predicted enzymes for converting preQ 1 to Q, would be expected to salvage the queuine (q) base, mirroring the scenario for the obligate intracellular pathogen Chlamydia trachomatis . However, sequence analyses indicate that the substrate-specificity residues of their bTGTs resemble those of enzymes inserting preQ 1 rather than q. Intriguingly, MS analyses of tRNA modification profiles in B. henselae reveal trace amounts of preQ 1 , previously not observed in a natural context. Complementation analysis demonstrates that B. henselae bTGT and QPTR not only utilize preQ 1 , akin to their Escherichia coli counterparts, but can also process q when provided at elevated concentrations. The experimental and phylogenomic analyses suggest that the Q pathway in B. henselae could represent an evolutionary transition among intracellular pathogens - from ancestors that synthesized Q de novo to a state prioritizing the salvage of q. Another possibility that will require further investigations is that the insertion of preQ 1 confers fitness advantages when B. henselae is growing outside a mammalian host.