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ABSTRACT Pseudomonas aeruginosais an opportunistic pathogen that can salvage nucleobases from the environment to conserve nutrients that would otherwise be spent onde novonucleotide biosynthesis. However, little is known regarding the substrate specificity of the 13 putative nucleobase transporters inP. aeruginosa. Here, using a combination of genetic and chemical approaches, we report substrate identifications for 10 putative nucleobase transporters inP. aeruginosa. Specifically, we individually expressed each transporter in a genetic background lacking all 13 putative nucleobase transporters and quantified growth on a panel of 10 nucleobases as sole nitrogen sources. We confirmed these expression-based substrate identifications using targeted genetic knockouts. In a complementary approach, we utilized four toxic nucleobase antimetabolites to characterize antimicrobial activity in these same strains. We identified the sole allantoin transporter as well as transporters for guanine, xanthine, uric acid, cytosine, thymine, uracil, and dihydrouracil. Furthermore, we associated at least five nucleobase transporters with hypoxanthine, which has been recently reported to be an antibiofilm cue inP. aeruginosa. These results provide an initial characterization of the putative nucleobase transporters inP. aeruginosa, significantly advancing our understanding of nucleobase transport in this clinically relevant organism. IMPORTANCEPseudomonas aeruginosais a frequently multidrug-resistant opportunistic pathogen and one of the most common causes of healthcare-acquired infections. While nucleobases are known to support growth in nutrient-limited conditions, recent work showed that adenine and hypoxanthine can also decreaseP. aeruginosabiofilm formation by disrupting c-di-GMP metabolism. Thus, nucleobase transport may be relevant to multiple aspects ofP. aeruginosabiology and pathogenesis. However, there is currently little known about the transport of nucleobases inP. aeruginosa. Our work reports initial substrate identifications for 10 putative nucleobase transporters inP. aeruginosa, providing new tools to address previously difficult-to-test hypotheses relating to nucleobase transport in this organism.