The obligate intracytoplasmic pathogen Rickettsia prowazekii relies on the transport of many essential compounds from the cytoplasm of the eukaryotic host cell in lieu of de novo synthesis, an evolutionary outcome undoubtedly linked to obligatory growth in this metabolite-replete niche. The paradigm for the study of rickettsial transport systems is the ATP/ADP translocase Tlc1, which exchanges bacterial ADP for host cell ATP as a source of energy, rather than as a source of adenylate. Interestingly, the R. prowazekii genome encodes four open reading frames that are highly homologous to the well-characterized ATP/ADP translocase Tlc1. Therefore, by annotation, the R. prowazekii genome encodes a total of five ATP/ADP translocases: Tlc1, Tlc2, Tlc3, Tlc4, and Tlc5. We have confirmed by quantitative reverse transcriptase PCR that mRNAs corresponding to all five tlc homologues are expressed in R. prowazekii growing in L-929 cells and have shown their heterologous protein expression in Escherichia coli, suggesting that none of the tlc genes are pseudogenes in the process of evolutionary meltdown. However, we demonstrate by heterologous expression in E. coli that only Tlc1 functions as an ATP/ADP transporter. A survey of nucleotides and nucleosides has determined that Tlc4 transports CTP, UTP, and GDP. Intriguingly, although GTP was not transported by Tlc4, it was an inhibitor of CTP and UTP uptake and demonstrated a K i similar to that of GDP. In addition, we demonstrate that Tlc5 transports GTP and GDP. We postulate that Tlc4 and Tlc5 serve the primary function of maintaining intracellular pools of nucleotides for rickettsial nucleic acid biosynthesis and do not provide the cell with nucleoside triphosphates as an energy source, as is the case for Tlc1. Although heterologous expression of Tlc2 and Tlc3 was observed in E. coli, we were unable to identify substrates for these proteins.Rickettsia prowazekii is the etiological agent of epidemic typhus in humans and is designated as a select agent based on its potential use as an agent of bioterrorism. R. prowazekii is an obligate intracellular bacterium and grows only within the cytoplasm of a eukaryotic host cell, unbounded by a host-derived membrane vesicle. As a consequence of growth in this nutrient-rich niche, R. prowazekii has evolved several unique transport systems specific for large, charged metabolites, such as UDP-glucose, S-adenosylmethionine, ATP, NAD, UMP, GMP, and AMP (11,12,45,52,54,55). The ability to transport substrates that are present in the host cell cytosol and rarely available in the extracellular milieu has likely contributed to the evolution of the small-sized rickettsial genome (834 open reading frames [ORFs]); many of the de novo biosynthetic pathways characteristic of free-living bacteria are no longer present in rickettsiae (8). The ATP/ADP translocase Tlc1 is the most thoroughly characterized of these transport systems (1-5, 13, 14, 16, 18, 27, 36, 50, 53, 56). Tlc1 is an obligateexchange transport system that catalyzes the equilibration of...