Reduction of ribonucleotides by the obligate intracytoplasmic bacterium Rickettsia prowazekii.

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 ribonucleotides 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 Escherichia coli.     

On the nature of obligate intracellular symbiosis of rickettsiae — Rickettsia prowazekii cells import mitochondrial porin

Mitochondrial porin was identified in Rickettsia prowazekii by Western blot analysis of whole cells and membrane fractions with monoclonal antibody against porin VDAC 1 of animal mitochondria. Using the BLAST server, no protein sequences homologous to mitochondrial porin were found among the rickettsial genomes. Rickettsiae also do not contain their own porin. The protein imported by rickettsiae is weakly extracted by nonionic detergents and, like porin in mitochondria, is insensitive to proteinase K in whole cells. Immunocytochemical analysis showed that it localizes to the outer membrane of the bacterial cells. These data support an earlier suggestion about import by rickettsiae of indispensable proteins from cytoplasm of the host cell as a molecular basis of obligate intracellular parasitism. They are also consistent with the hypothesis invoking a transfer of genes specifying surface proteins from the last common ancestor of rickettsiae and mitochondria to the host genome, and preservation by rickettsiae of the primitive ability to import these proteins.     

S-adenosylmethionine transport in Rickettsia prowazekii

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 micro M. Transport was inhibited by S-adenosylethionine and S-adenosylhomocysteine 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.     

Acquisition of polyamines by the obligate intracytoplasmic bacterium Rickettsia prowazekii.

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 10(8) 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 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.     

Analysis of the peptidoglycan of Rickettsia prowazekii.

In the present study, peptidoglycan from Rickettsia prowazekii, an obligate intracellular bacterium, was purified. The rickettsial peptidoglycan is like that of gram-negative bacteria; that is, it is sodium dodecyl sulfate insoluble, lysozyme sensitive, and composed of glutamic acid, alanine, and diaminopimelic acid in a molar ratio of 1.0:2.3:1.0. The small amount of lysine found in the peptidoglycan preparation suggests that a peptidoglycan-linked lipoprotein(s) may be present in the rickettsiae. D-Cycloserine, a D-alanine analog which inhibits the biosynthesis of bacterial cell walls, prevented rickettsial growth in mouse L929 cells at a high concentration and altered the morphology of the rickettsiae at a low concentration. These effects were prevented by the addition of D-alanine. This suggests that R. prowazekii contains D-alanine in the peptidoglycan and has D-Ala-D-Ala ligase and alanine racemase activities.     

Complete genome sequence of Rickettsia typhi and comparison with sequences of other rickettsiae

Rickettsia typhi, the causative agent of murine typhus, is an obligate intracellular bacterium with a life cycle involving both vertebrate and invertebrate hosts. Here we present the complete genome sequence of R. typhi (1,111,496 bp) and compare it to the two published rickettsial genome sequences: R. prowazekii and R. conorii. We identified 877 genes in R. typhi encoding 3 rRNAs, 33 tRNAs, 3 noncoding RNAs, and 838 proteins, 3 of which are frameshifts. In addition, we discovered more than 40 pseudogenes, including the entire cytochrome c oxidase system. The three rickettsial genomes share 775 genes: 23 are found only in R. prowazekii and R. typhi, 15 are found only in R. conorii and R. typhi, and 24 are unique to R. typhi. Although most of the genes are colinear, there is a 35-kb inversion in gene order, which is close to the replication terminus, in R. typhi, compared to R. prowazekii and R. conorii. In addition, we found a 124-kb R. typhi-specific inversion, starting 19 kb from the origin of replication, compared to R. prowazekii and R. conorii. Inversions in this region are also seen in the unpublished genome sequences of R. sibirica and R. rickettsii, indicating that this region is a hot spot for rearrangements. Genome comparisons also revealed a 12-kb insertion in the R. prowazekii genome, relative to R. typhi and R. conorii, which appears to have occurred after the typhus (R. prowazekii and R. typhi) and spotted fever (R. conorii) groups diverged. The three-way comparison allowed further in silico analysis of the SpoT split genes, leading us to propose that the stringent response system is still functional in these rickettsiae.     

Potassium permeability of Rickettsia prowazekii.

The potassium permeability of Rickettsia prowazekii was characterized by chemical measurement of the intracellular sodium and potassium pools and isotopic flux measurements with 86Rb+ as a tracer. R. prowazekii, in contrast to Escherichia coli, did not maintain a high potassium-to-sodium ratio in their cytoplasm except when the potassium-to-sodium ratio in the extracellular medium was high or when the extracellular concentrations of both cations were low (ca. 1 mM). Both influx and efflux assays with 86Rb+ demonstrated that the rickettsial membrane had limited permeability to potassium and that incorporation of valinomycin into these cells increased these fluxes at least 10-fold. The transport of potassium showed specificity and dependence on rickettsial metabolism. The increased flux of potassium which results from the incorporation of valinomycin into the rickettsial membrane was detrimental to both lysine transport and lysis of erythrocytes by the rickettsiae.     

Phospholipid composition of Rickettsia prowazeki grown in chicken embryo yolk sacs.

The phospholipid composition and phospholipid fatty acid composition of purified Rickettsia prowazeki were determined. The lipid phosphorous content was 6.8 +/- 1.3 microgram/mg of total rickettsial protein. The major phospholipid was phosphatidylethanolamine (60 to 70%); phosphatidylglycerol constituted 20%, and phosphatidylcholine constituted 15%. Small amounts of phosphatidylserine and cardiolipin were detected. The principal fatty acids were 18:1, 16:1, and 16:0. The fatty acid composition of the phosphatidylcholine in the rickettsial extracts was very different than that of the other rickettsial phosphatides and very similar to that of normal yolk sac phosphatidylcholine. The specific of the phosphatidylcholine of rickettsiae grown in the presence of 32P was markedly lower than that of phosphatidylethanolamine and phosphatidylglycerol. It is suggested that the phosphatidylcholine in the rickettsial extract is yolk sac derived and either tightly absorbed or exchanged into the rickettsial membrane.     

Purification and partial characterization of the DNA-dependent RNA polymerase from Rickettsia prowazekii

The DNA-dependent RNA polymerase was purified from Rickettsia prowazekii, an obligate intracellular bacterial parasite. Because of limitation of available rickettsiae, the classical methods for isolation of the enzyme from other procaryotes were modified to purify RNA polymerase from small quantities of cells (25 mg of protein). The subunit composition of the rickettsial RNA polymerase was typical of a eubacterial RNA polymerase. R. prowazekii had beta' (148,000 daltons), beta (142,000 daltons), sigma (85,000 daltons), and alpha (34,500 daltons) subunits as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The appropriate subunits of the rickettsial RNA polymerase bound to polyclonal antisera against Escherichia coli core polymerase and E. coli sigma 70 subunit in Western blots (immunoblots). The enzyme activity was dependent on all four ribonucleoside triphosphates, Mg2+, and a DNA template. Optimal activity occurred in the presence of 10 mM MgCl2 and 50 mM NaCl. Interestingly, in striking contrast to E. coli, approximately 74% of the rickettsial RNA polymerase activity was associated with the rickettsial cell membrane at a low salt concentration (50 mM NaCl) and dissociated from the membrane at a high salt concentration (600 mM NaCl).     

Rickettsia prowazekii Transports UMP and GMP, but Not CMP, as Building Blocks for RNA Synthesis

Rickettsia prowazekii, the etiological agent of epidemic typhus, is an obligate intracellular bacterium and is apparently unable to synthesize ribonucleotides de novo. Here, we show that as an alternative, isolated, purified R. prowazekii organisms transported exogenous uridyl- and guanylribonucleotides and incorporated these labeled precursors into their RNA in a rifampin-sensitive manner. Transport systems for nucleotides, which we have shown previously and show here are present in rickettsiae, have never been reported in free-living bacteria, and the usual nucleobase and nucleoside transport systems are absent in rickettsiae. There was a clear preference for the monophosphate form of ribonucleotides as the transported substrate. In contrast, rickettsiae did not transport cytidylribonucleotides. The source of rickettsial CTP appears to be the transport of UMP followed by its phosphorylation and the amination of intrarickettsial UTP to CTP by CTP synthetase. A complete schema of nucleotide metabolism in rickettsiae is presented that is based on a combination of biochemical, physiological, and genetic information.     

Rickettsial metK-encoded methionine adenosyltransferase expression in an Escherichia coli metK deletion strain

The obligate intracellular bacterium Rickettsia prowazekii has recently been shown to transport the essential metabolite S-adenosylmethionine (SAM). The existence of such a transporter would suggest that the metK gene, coding for the enzyme that synthesizes SAM, is unnecessary for rickettsial growth. Genome sequencing has revealed that this is the case for the metK genes of the spotted fever group and the Madrid E strain of R. prowazekii, which contain recognizable inactivating mutations. However, several strains of the typhus group rickettsiae possess metK genes lacking obvious mutations. In order to determine if these genes code for a product that retains MAT function, an Escherichia coli metK deletion mutant was constructed in which individual rickettsial metK genes were tested for the ability to complement the methionine adenosyltransferase deficiency. Both the R. prowazekii Breinl and R. typhi Wilmington metK genes complemented at a level comparable to that of an E. coli metK control, demonstrating that the typhus group rickettsiae have the capability of synthesizing as well as transporting SAM. However, the appearance of mutations that affect the function of the metK gene products (a stop codon in the Madrid E strain and a 6-bp deletion in the Breinl strain) provides experimental support for the hypothesis that these typhus group genes, like the more degenerate spotted fever group orthologs, are in the process of gene degradation.     

Transposon mutagenesis of the obligate intracellular pathogen Rickettsia prowazekii

Genetic analysis of Rickettsia prowazekii has been hindered by the lack of selectable markers and efficient mechanisms for generating rickettsial gene knockouts. We have addressed these problems by adapting a gene that codes for rifampin resistance for expression in R. prowazekii and by incorporating this selection into a transposon mutagenesis system suitable for generating rickettsial gene knockouts. The arr-2 gene codes for an enzyme that ADP-ribosylates rifampin, thereby destroying its antibacterial activity. Based on the published sequence, this gene was synthesized by PCR with overlapping primers that contained rickettsial codon usage base changes. This R. prowazekii-adapted arr-2 gene (Rparr-2) was placed downstream of the strong rickettsial rpsL promoter (rpsL(P)), and the entire construct was inserted into the Epicentre EZ::TN transposome system. A purified transposon containing rpsL(P)-Rparr-2 was combined with transposase, and the resulting DNA-protein complex (transposome) was electroporated into competent rickettsiae. Following selection with rifampin, rickettsiae with transposon insertions in the genome were identified by PCR and Southern blotting and the insertion sites were determined by rescue cloning and inverse PCR. Multiple insertions into widely spaced areas of the R. prowazekii genome were identified. Three insertions were identified within gene coding sequences. Transposomes provide a mechanism for generating random insertional mutations in R. prowazekii, thereby identifying nonessential rickettsial genes.     

Proteome analysis of Madrid E strain of Rickettsia prowazekii

Rickettsia prowazekii, an obligate intracellular Gram-negative bacterium, is the etiologic agent of epidemic typhus. The threat of typhus as a biological weapon lies in its stability in the dried louse feces and in its infection by inhalation of an aerosol. Consequently, it is listed as a select agent and warrants more research to understand its pathogenesis. Although the genomic DNA sequence of strain Madrid E has been completed, the actual expression of the individual protein has not been investigated. In order to provide a global view of the expressed protein profile, the whole cell lysate of purified rickettsia (Madrid E strain) was reduced, alkylated, and digested with trypsin. The total digest was characterized by a two-dimensional liquid chromatography mass spectrometry system and analyzed with a modified version of the ProteomeX workstation. A total of 252 proteins out of 834 predicted protein-coding genes were identified, 238 proteins were identified by the detection of at least two unique peptides. Only 14 proteins were identified by the detection of one unique peptide in all three separate analyses. Among the 238 proteins identified by multiple unique peptides, 230 proteins were found in at least two of three separate analyses. The reproducible and convenient methodology and the information described here have provided a foundation for future proteome study of various R. prowazekii strains with different virulence.     

Acquisition of thymidylate by the obligate intracytoplasmic bacterium Rickettsia prowazekii.

The pathway for the acquisition of thymidylate in the obligate bacterial parasite Rickettsia prowazekii was determined. R. prowazekii growing in host cells with or without thymidine kinase failed to incorporate into its DNA the [3H]thymidine added to the culture. In the thymidine 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, [3H]uridine incorporation into the DNA of R. prowazekii was demonstrable (973 +/- 57 dpm/3 x 10(8) 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. prowazekii. The enzyme was determined to have a specific activity of 310 +/- 40 pmol/min/mg of protein and was inhibited greater than or equal to 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 (Urd-A) supplemented with 50 microM to 1 mM uracil. In contrast, the rickettsiae were able to grow in Urd-A cells that were uridine starved and supplemented with 20 microM uridine (117% of control). However, no measurable uridine kinase activity could be measured 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.     

Some lessons from Rickettsia genomics

Sequencing of the Rickettsia conorii genome and its comparison with its closest sequenced pathogenic relative, i.e., Rickettsia prowazekii, provided powerful insights into the evolution of these microbial pathogens. However, advances in our knowledge of rickettsial diseases are still hindered by the difficulty of working with strict intracellular bacteria and their hosts. Information gained from comparing the genomes of closely related organisms will shed new light on proteins susceptible to be targeted in specific diagnostic assays, by new antimicrobial drugs, and that could be employed in the generation of future rickettsial vaccines. In this review we present a detailed comparison of the metabolic pathways of these bacteria as well as the polymorphisms of their membrane proteins, transporters and putative virulence factors. Environmental adaptation of Rickettsia is also discussed.     

Propagation of arthropod-borne Rickettsia spp. in two mosquito cell lines

Rickettsiae are obligate intracellular alphaproteobacteria that include pathogenic species in the spotted fever, typhus, and transitional groups. The development of a standardized cell line in which diverse rickettsiae can be grown and compared would be highly advantageous to investigate the differences among and between pathogenic and nonpathogenic species of rickettsiae. Although several rickettsial species have been grown in tick cells, tick cells are more difficult to maintain and they grow more slowly than insect cells. Rickettsia-permissive arthropod cell lines that can be passaged rapidly are highly desirable for studies on arthropod-Rickettsia interactions. We used two cell lines (Aedes albopictus cell line Aa23 and Anopheles gambiae cell line Sua5B) that have not been used previously for the purpose of rickettsial propagation. We optimized the culture conditions to propagate one transitional-group rickettsial species (Rickettsia felis) and two spotted-fever-group rickettsial species (R. montanensis and R. peacockii) in each cell line. Both cell lines allowed the stable propagation of rickettsiae by weekly passaging regimens. Stable infections were confirmed by PCR, restriction digestion of rompA, sequencing, and the direct observation of bacteria by fluorescence in situ hybridization. These cell lines not only supported rickettsial growth but were also permissive toward the most fastidious species of the three, R. peacockii. The permissive nature of these cell lines suggests that they may potentially be used to isolate novel rickettsiae or other intracellular bacteria. Our results have important implications for the in vitro maintenance of uncultured rickettsiae, as well as providing insights into Rickettsia-arthropod interactions.     

Transformation of Rickettsia prowazekii to erythromycin resistance encoded by the Escherichia coli ereB gene

Rickettsia prowazekii, the etiologic agent of epidemic typhus, is an obligate, intracytoplasmic, parasitic bacterium. Recently, the transformation of this bacterium via electroporation has been reported. However, in these studies identification of transformants was dependent upon either selection of an R. prowazekii rpoB chromosomal mutation imparting rifampin resistance or expression of the green fluorescent protein and flow cytometric analysis. In this paper we describe the expression in R. prowazekii of the Escherichia coli ereB gene. This gene codes for an erythromycin esterase that cleaves erythromycin. To the best of our knowledge, this is the first report of the expression of a nonrickettsial, antibiotic-selectable gene in R. prowazekii. The availability of a positive selection for rickettsial transformants is an important step in the characterization of genetic analysis systems in the rickettsiae.     

The nucleotide transporter of Caedibacter caryophilus exhibits an extended substrate spectrum compared to the analogous ATP/ADP translocase of Rickettsia prowazekii

The two obligate intracellular alphaproteobacteria Rickettsia prowazekii and Caedibacter caryophilus, a human pathogen and a paramecium endosymbiont, respectively, possess transport systems to facilitate ATP uptake from the host cell cytosol. These transport proteins, which have 65% identity at the amino acid level, were heterologously expressed in Escherichia coli, and their properties were compared. The results presented here demonstrate that the caedibacter transporter had a broader substrate than the more selective rickettsial transporter. ATP analogs with modified sugar moieties, dATP and ddATP, inhibited the transport of ATP by the caedibacter transporter but not by the rickettsial transporter. Both transporters were specific for di- and trinucleotides with an adenine base in that adenosine tetraphosphate, AMP, UTP, CTP, and GTP were not competitive inhibitors. Furthermore, the antiporter nature of both transport systems was shown by the dependence of the efflux of [alpha-32P]ATP on the influx of substrate (ATP but not dATP for rickettsiae, ATP or dATP for caedibacter).     

Phylogenetic analysis based on groE shows the closest evolutionary relationship between mitochondria and Rickettsia]

The nucleotide sequence of the groE operon of Rickettsia prowazekii, the obligate intracellular parasite of eukaryotes, was determined. The alignment of DNA-inferred amino acid sequences of the Hsp10 and Hsp60 heat-shock proteins with bacterial and mitochondrial homologues revealed the presence within Hsp60 of signatures shared by mitochondria and rickettsiae. Phylogenetic analysis demonstrated that heat-shock proteins of R. prowazekii are the earliest and the least diverging homologues within the family Rickettsiaceae--a sister group to the monophyletic clade of mitochondria. These results are in good agreement with the data obtained when using other molecular chronometers and show the closest relationship between mitochondria and Rickettsia. The possible nature of obligate intracellular parasitism of rickettsiae has been considered on the basis of the assumption that they and mitochondria could have a common evolutionary origin.