We describe here the complete genome sequence (1,111,523 base pairs) of the obligate intracellular parasite Rickettsia prowazekii, the causative agent of epidemic typhus. This genome contains 834 protein-coding genes. The functional pro®les of these genes show similarities to those of mitochondrial genes: no genes required for anaerobic glycolysis are found in either R. prowazekii or mitochondrial genomes, but a complete set of genes encoding components of the tricarboxylic acid cycle and the respiratory-chain complex is found in R. prowazekii. In effect, ATP production in Rickettsia is the same as that in mitochondria. Many genes involved in the biosynthesis and regulation of biosynthesis of amino acids and nucleosides in free-living bacteria are absent from R. prowazekii and mitochondria. Such genes seem to have been replaced by homologues in the nuclear (host) genome. The R. prowazekii genome contains the highest proportion of non-coding DNA (24%) detected so far in a microbial genome. Such non-coding sequences may be degraded remnants of`neutralized' genes that await elimination from the genome. Phylogenetic analyses indicate that R. prowazekii is more closely related to mitochondria than is any other microbe studied so far.The Rickettsia are a-proteobacteria that multiply in eukaryotic cells only. R. prowazekii is the agent of epidemic, louse-borne typhus in humans. Three features of this endocellular parasite deserve our attention. First, R. prowazekii is estimated to have infected 20±30 million humans in the wake of the First World War and killed another few million following the Second World War (ref. 1). Because it is the descendent of free-living organisms 2±4 , its genome provides insight into adaptations to the obligate intracellular lifestyle, with probable practical value. Second, phylogenetic analyses based on sequences of ribosomal RNA and heat-shock proteins indicate that mitochondria may be derived from the aproteobacteria 5,6 . Indeed, the closest extant relatives of the ancestor to mitochondria seem to be the Rickettsia 7±10 . That modern Rickettsia favour an intracellular lifestyle identi®es these bacteria as the sort of organism that might have initiated the endosymbiotic scenario leading to modern mitochondria 11 . Finally, the genome of R. prowazekii is a small one, containing only 1,111,523 base pairs (bp). Its phylogenetic placement and many other characteristics identify it as a descendant of bacteria with substantially larger genomes 2±4 . Thus Rickettsia, like mitochondria, are good examples of highly derived genomes, the products of several types of reductive evolution.The genome sequence of R. prowazekii indicates that these three features may be related. For example, prokaryotic genomes evolving within a cell dominated by a much larger, eukaryote genome and constrained by bottle-necked population dynamics will tend to lose genetic information 12,13 . Predictable sets of expendable genes will tend to disappear from the prokaryotic genome when they are made redundant by the activit...

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Cosmid clone banks of Rickettsia prowazekii genomic DNA were established in Escherichia coli and screened for expression of the rickettsial carrier-mediated ADP/ATP translocator. Out of 2700 clones screened, a single clone, designated MOB286, accumulated radioactivity when incubated with [a-32P] Uptake of ATP was also temperature-dependent, insensitive to atractyloside, N-ethylmaleimide, and dinitrophenol, and specific for ADP and ATP. Efflux of radiolabeled nucleotide was observed in the presence of extracellular ADP or ATP but not AMP and was not observed in the absence of extracellular adenine nucleotides. The successful cloning and expression of the rickettsial ADP/ATP translocator in E. coli will permit better characterization of rickettsial bioenergetics and of the metabolic regulation of obligate intracellular parasitism.Rickettsia prowazekii, the etiologic agent of epidemic typhus, is an obligate intracellular parasite. Whereas other intracellular bacteria are generally found surrounded by a membrane of host origin, rickettsiae escape quickly from phagocytic vacuoles and grow directly within the cytoplasm (and occasionally the nucleus) of their host cell. Rickettsiae are not leaky, as had been postulated, but possess both usual and unusual transport systems (1-4), most notably, a carriermediated ADP/ATP translocator (1). This activity, similar to that present in mitochondria, permits the exchange of ADP from the rickettsia for ATP present in the host cell's cytoplasm. The carrier is specific for ADP and ATP, operates equally well in either direction, has no energy requirement, and is of an obligatory exchange type-i.e., for every molecule of ATP transported into a rickettsia, a molecule of ADP must be transported from the rickettsia, or vice versa. ADP/ ATP exchange is regulated at least in part by the concentration of inorganic phosphate (5).Characterization of the ADP/ATP translocator is fundamental to gaining an understanding of rickettsial physiology and the regulation thereof. This task is complicated enormously by the difficulty in obtaining large quantities of highly purified rickettsiae due to the required intracellular propagation of these bacteria, usually in embryonated hen eggs. Furthermore, attempts to form membrane vesicles from R. prowazekii, which would be very important in this characterization, have been unsuccessful (unpublished observation). The cloning and expression in Escherichia coli of the gene(s) responsible for ADP/ATP exchange would permit circumvention of these problems. Purification of the translocator would be facilitated immensely, modulation of this activity by various effectors could be studied against a welldefined E. coli physiological background, membrane vesicles could be formed, and gene organization and regulation could be assessed. We report here the cloning in E. coli of a R. prowazekii genetic locus that directs the expression in E. coli of the rickettsial ADP/ATP translocator.

Rickettsia prowazekii, the causative agent of epidemic typhus, is an obligate, intracellular, parasitic bacterium that grows within the cytoplasm of eucaryotic host cells. Rickettsiae exploit this intracellular environment by using transport systems for the compounds available in the host cell's cytoplasm. Analysis of the R. prowazekii Madrid E genome sequence revealed the presence of a mutation in the rickettsial metK gene, the gene encoding the enzyme responsible for the synthesis of S-adenosylmethionine (AdoMet). Since AdoMet is required for rickettsial processes, the apparent inability of this strain to synthesize AdoMet suggested the presence of a rickettsial AdoMet transporter. We have confirmed the presence of an AdoMet transporter in the rickettsiae which, to our knowledge, is the first bacterial AdoMet transporter identified. The influx of AdoMet into rickettsiae was a saturable process with a K T of 2.3 M. Transport was inhibited by S-adenosylethionine and Sadenosylhomocysteine but not by sinfungin or methionine. Transport was also inhibited by 2,4-dinitrophenol, suggesting an energy-linked transport mechanism, and by N-ethylmaleimide. AdoMet transporters with similar properties were also identified in the Breinl strain of R. prowazekii and in Rickettsia typhi. By screening Escherichia coli clone banks for AdoMet transport, the R. prowazekii gene coding for a transporter, RP076 (sam), was identified. AdoMet transport in E. coli containing the R. prowazekii sam gene exhibited kinetics similar to that seen in rickettsiae. The existence of a rickettsial transporter for AdoMet raises intriguing questions concerning the evolutionary relationship between the synthesis and transport of this essential metabolite.

The amino acid sequence of the proposed glucose-6-phosphate (Glc6P) transporter from Chlamydia pneumoniae (HPTcp; hexose phosphate transporter [Chlamydia pneumoniae]) exhibits a higher degree of similarity to the Escherichia coli Glc6P sensor (UhpC) than to the E. coli Glc6P transporter (UhpT). Overexpression of His-UhpC in a UhpT-deficient E. coli strain revealed that the sensor protein is also able to transport Glc6P and exhibits an apparent K m (Glc6P) of 25 M, whereas His-HPTcp exhibits an apparent K m (Glc6P) of 98 M. His-HPTcp showed a four-times-lower specific activity than His-UhpT but a 56-times-higher specific activity than His-UhpC. Like His-UhpT and His-UhpC, the carrier His-HPTcp performs a sugar-phosphate/inorganicphosphate antiporter mode of transport. Surprisingly, while physiological concentrations of inorganic phosphate competitively inhibited transport mediated by the E. coli proteins His-UhpT and His-UhpC, transport mediated by His-HPTcp was not inhibited. Interestingly, C 3 -organophosphates stimulated His-HPTcp activity but not His-UhpT-or His-UhpC-catalyzed Glc6P transport. In contrast to His-UhpC, the His-HPTcp protein does not act as a Glc6P sensor in the uhp regulon.