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ABSTRACTThe phagosomal transporter (Pht) family of the major facilitator superfamily (MFS) is encoded by phylogenetically related intracellular gammaproteobacteria, including the opportunistic pathogen Legionella pneumophila. The location of the phtgenes between the putative thymidine kinase (tdk) and phosphopentomutase (deoB) genes suggested that the phtCand phtDloci contribute to thymidine salvage in L. pneumophila. Indeed, a phtC+allele in transrestored pyrimidine uptake to an Escherichia colimutant that lacked all known nucleoside transporters, whereas a phtD+allele did not. The results of phenotypic analyses of L. pneumophilastrains lacking phtCor phtDstrongly indicate that L. pneumophilarequires PhtC and PhtD function under conditions where sustained dTMP synthesis is compromised. First, in broth cultures that mimicked thymidine limitation or starvation, L. pneumophilaexhibited a marked requirement for PhtC function. Conversely, mutation of phtDconferred a survival advantage. Second, in medium that lacked thymidine, multicopy phtC+or phtD+alleles enhanced the survival of L. pneumophilathymidylate synthase (thyA)-deficient strains, which cannot synthesize dTMP endogenously. Third, under conditions in which transport of the pyrimidine nucleoside analog 5-fluorodeoxyuridine (FUdR) would inhibit growth, PhtC and PhtD conferred a growth advantage to L. pneumophilathyA+strains. Finally, when cultured in macrophages, L. pneumophilarequired the phtC-phtDlocus to replicate. Accordingly, we propose that PhtC and PhtD contribute to protect L. pneumophilafrom dTMP starvation during its intracellular life cycle.

The specificity of the ATP:corrinoid adenosyltransferase (CobA) enzyme of Salmonella entericaserovar Typhimurium LT2 for its nucleotide substrate was tested using ATP analogs and alternative nucleotide donors. The enzyme showed broad specificity for the nucleotide base and required the 2′-OH group of the ribosyl moiety of ATP for activity. 31P NMR spectroscopy was used to identify inorganic triphosphate (PPPi) as the byproduct of the reaction catalyzed by the CobA enzyme. Cleavage of triphosphate into pyrophosphate and orthophosphate did not occur, indicating that triphosphate cleavage was not required for release of the adenosylcorrinoid product. Triphosphate was a strong inhibitor of the reaction, with 85% of CobA activity lost when the ATP/PPPiratio present in the reaction mixture was 1:2.5.