The pathway for the acquisition of thymidylate in the obligate bacterial parasite RickeUtsia prowazekii was determined. R. prowazekii growing in host cells with or without thymidine kinase failed to incorporate into its DNA the [3Hlthymidine added to the culture. In the thyiiidlne kinase-negative host cells, the label available to the rickettsiae in the host cell cytoplasm would have been thymidine, and in the thymidine kinase-positive host cells, it would have been both thymidine and TMP. Further support for the inability to utilize thymidine was the lack of thymidine kinase activity in extracts of R. prowazekii. However, [3ljuridine incorporation into the DNA of R. prowazekii was demonstrable (973 ± 57 dpm/3 x 108 rickettsiae). This labeling of rickettsial DNA suggests the transport of uracil, uridine, uridine phosphates (UXP), or 2'-deoxyuridine phosphates, the conversion of the labeled precursor to thymidylate, and subsequent incorporation into DNA. This is supported by the demonstration of thymidylate synthase activity in extracts of R. prowazeku. The enzyme was determined to have a specific activity of 310 ± 40 pmol/min/mg of protein and was inhibited .70% by 5-fluoro-dUMP. The inability of R. prowazekii to utilize uracil was suggested by undetectable uracil phosphoribosyltransferase activity and by its inability to grow (less than 10% of control) in a uridine-starved mutant cell line (UrdFA) supplemented with 50 pM'to 1 mM uracil. In contrast, the rickettsiae were able to grow in Urd-A cells that were uridine starved and supplemented with 20 ,uM uridine (117% of control). However, no measurable uridine kinase activity could be measur'ed in extracts of R. prowazekii. Normal rickettsial growth (92% of control) was observed when the host. cell was blocked with thymidine so that the host cell's dUXP pool was depressed to a level inadequate for growth and DNA synthesis in the host cell. Taken together, these data strongly suggest that rickettsiae transport UXP from the host cell's cytoplasm and that they synthesize TTP from UXP.
Both the polyamine content and the route of acquisition of polyamines by Rickettsia prowazekii, an obligate intracellular parasitic bacterium, were determined. The rickettsiae grew normally in an ornithine decarboxylase mutant of the Chinese hamster ovary (C55.7) cell line whether or not putrescine, which this host cell required in order to grow, was present. The rickettsiae contained approximately 6 mM putrescine, 5 mM spermidine, and 3 mM spermine when cultured in the presence or absence of putrescine. Neither the transport of putrescine and spermidine by the rickettsiae nor a measurable rickettsial ornithine decarboxylase activity could be demonstrated. However, we demonstrated the de novo synthesis of polyamines from arginine by the rickettsiae. Arginine decarboxylase activity (29 pmol of 14Co2 released per h per 108 rickettsiae) was measured in the rickettsiae growing within their host cell. A markedly lower level of this enzymatic activity was observed in cell extracts of R. prowazekii and could be completely inhibited with 1 mM difluoromethylarginine, an irreversible inhibitor of the enzyme. R. prowazekii failed to grow in C55.7 cells that had been cultured in the presence of 1 mM difluoromethylarginine. After rickettsiae were grown in C55.7 cells in the presence of labeled arginine, the specific activities of arginine in the host cell cytoplasm and polyamines in the rickettsiae were measured; these measurements indicated that 100% of the total polyamine content of R. prowazekii was derived from arginine.Rickettsia prowazekii, the etiological agent of epidemic typhus, is an obligate intracytoplasmic bacterium. R. prowazekii grows free in the cytoplasm of its eucaryotic host cell unbounded by any internal membranes of host cell origin. If the rickettsiae have evolved appropriate transport systems, then they would be able to scavenge from this rich external milieu many of the end products and intermediate metabolites needed for growth rather than expending energy synthesizing these metabolites via complex pathways. For example, the enzymes of the glycolytic and hexose monophosphate pathways are absent in typhus group rickettsiae (9) and the essential metabolites generated by these pathways must be obtained from the cytoplasm of the host cell. Rickettsiae can also employ a dual strategy for the acquisition of metabolites: they can both transport and synthesize the same compound. This can be seen most clearly in the acquisition of ATP. The enzymes of the tricarboxylic acid cycle (9), oxidative phosphorylation (7, 12), and an ATPase (37) are present in the rickettsiae to synthesize ATP. However, the rickettsiae also have a transport system to obtain ATP from the cytoplasm of the host cell (32).Virtually all cells contain significant amounts of some or all of the polyamines putrescine, spermidine, and spermine. Proposed functions for these highly basic amines include, but are not limited to, the stabilization of nucleic acids and the stimulation of protein biosynthesis in both eucaryotic and procaryotic organis...
PepA with the characterized leucine aminopeptidases from E. coli, Arabidopsis thaliana, and bovine eye lens revealed that 39.8, 34.9, and 34.0% of the residues were identical, respectively. Residues proposed to be part of the active site or involved in the binding of metal ions in the bovine metalloenzyme were all conserved in R. prowazekii PepA. However, despite the structural and enzymatic similarity to E. coli PepA, the R. prowazekii protein was unable to complement the cer site-specific, PepA-dependent recombination system found in E. coli that resolves ColEl-type plasmid multimers into their monomeric forms.
All currently available assays for antibodies to the etiologic agents of spotted fever group rickettsioses detect reactivity with antigens of lipopolysaccharides and the major cell wall proteins, which contain epitopes that are shared among many spotted fever group rickettsiae. The hypothesis of this study was that a monoclonal antibody to a species-specific epitope of Rickettsia rickettsii would be blocked from binding to the rickettsial surface if the rickettsiae were incubated previously with serum containing species-specific antibodies to the epitope. In an EIA, binding of monoclonal antibody 5C10-F3 to R. rickettsii was blocked by convalescent sera from patients with Rocky Mountain spotted fever but not from those with Mediterranean spotted fever, endemic typhus, or Q fever or normal subjects. This assay should be useful in determining the origin of the high seroprevalence of spotted fever rickettsial antibodies in certain geographic regions and establishing a species-specific diagnosis in patients with undetermined rickettsial exposure.
Rickettsia prowazekii, an obligate intracellular parasitic bacterium, was shown to have a ribonucleotide reductase that would allow the rickettsiae to obtain the deoxyribonucleotides needed for DNA synthesis from rickettsial ribonudeotides rather than from transport. In the presence of hydroxyurea, R. prowazekii failed to grow in mouse L929 cells or SC2 cells (a hydroxyurea-resistant cell line), which suggested that R. prowazekii contains a functional ribonucleotide reductase. This enzymatic activity was demonstrated by the conversion of ADP to dADP and CDP to dCDP, using (i) a crude extract of Renografin-purified R. prowazekii that had been harvested from infected yolk sacs and (ii) high-performance liquid chromatographic analysis. The rickettsial ribonucleotide reductase utilized ribonucleoside diphosphates as substrates, required magnesium and a reducing agent, and was inhibited by hydroxyurea. ADP reduction was stimulated by dGTP and inhibited by dATP. CDP reduction was stimulated by ATP and adenylylimido-diphosphate and inhibited by dATP and dGTP. These characteristics provided strong evidence that the rickettsial enzyme is a nonheme iron-containing enzyme similar to those found in mammalian cells and aerobic Escherchia coli.Rickettsia prowazekii, the etiological agent of epidemic typhus in humans, is an obligate intracellular parasitic bacterium. The rickettsiae grow directly in the host cell's cytoplasm and are not bounded by phagosomal or phagolysosomal membranes (24). This unusual external milieu provides the rickettsiae with a rich source of preformed metabolites.Knowledge of the metabolism and transport activities of rickettsiae for nucleotides and related metabolites is limited. The route of acquisition of the deoxyribonucleotides needed for DNA synthesis is unknown. Two distinct possibilities exist in such an obligate intracytoplasmic bacterium. The rickettsiae could transport deoxyribonucleotides from the host cell's cytoplasm and incorporate them into their DNA directly. Alternatively, the rickettsiae could transport ribonucleotides and then synthesize deoxyribonucleotides by employing a rickettsial ribonucleotide reductase. The ability ofR. prowazekii to transport large, charged metabolites from the host cell's cytoplasm is well documented (26). For example, carrier-mediated membrane transport systems that provide for exchange of intra-and extracellular adenine nucleotides have been described in R. prowazekii for both AMP (1) and ATP/ADP (25). However, no system for the net transport of ribonucleotides, ribonucleosides, ribonucleobases, or the corresponding deoxy forms has been described.Ribonucleotide reductase (ribonucleoside diphosphate reductase [EC 1.17.4.1] and ribonucleoside triphosphate reductase [EC 1.17.4.2]) catalyzes the conversion of ribonucleotides to their corresponding deoxyribonucleotides (10,18,22). The activity of this enzymatic system is controlled by ribo-and deoxyribonucleotides, dithiols, and additional metal ions in complex ways. Because the pool size of deoxyribonucleosid...
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