A sulfur-containing nucleoside antibiotic from <i>Photorhabdus</i>

We report the discovery of 4'-thiothymidine (4'sT), a sulfur-containing nucleoside antibiotic produced by the nematode symbiont <i>Photorhabdus asymbiotica</i>. 4'sT exhibits antibacterial activity against <i>Escherichia coli</i>, <i>Stenotrophomonas maltophilia</i>, and <i>Klebsiella pneumoniae</i>. The production of 4'sT was increased in rifampicin-resistant mutants. Heterologous expression of the radical SAM enzyme-containing gene cluster confirmed its biosynthetic origin. Resistant mutants mapped to <i>tdk,</i> which encodes thymidine kinase in the thymidine salvage pathway. Mechanistic studies revealed that 4'sT is a prodrug activated via the thymidine salvage pathway and incorporated into DNA, where it inhibits replication and triggers the SOS response. Clinical <i>E. coli</i> isolates were resistant, but inhibition of <i>de novo</i> thymidylate synthesis with trimethoprim rendered them highly susceptible, indicating that strain-selective activity is governed by differences in thymidine metabolism. These findings highlight the role of metabolic context in determining antibiotic selectivity and demonstrate the utility of rifampicin resistance for assessing silent biosynthetic gene clusters to discover new antimicrobial compounds.IMPORTANCEIntroducing novel antibiotics is essential to counter the spread of drug-resistant pathogens. Here, we report the discovery of 4'-thiothymidine (4'sT), a nucleoside antibiotic from the nematode symbiont <i>Photorhabdus asymbiotica</i>, identified through activation of a silent biosynthetic gene cluster. 4'sT features an unusual 4'-thiosugar moiety. We identified its biosynthetic gene cluster, including a radical SAM enzyme presumably involved in sulfur incorporation. 4'sT exhibits strain-selective activity that is governed by differences in thymidine metabolism rather than variations in the molecular target. These findings expand our knowledge of antibiotics with unusual selective activity.