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.     

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.     

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.     

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.     

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).     

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.     

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.     

Mariner-based transposon mutagenesis of Rickettsia prowazekii

Rickettsia prowazekii, the causative agent of epidemic typhus, is an obligate intracellular bacterium that grows directly within the cytoplasm of its host cell, unbounded by a vacuolar membrane. The obligate intracytoplasmic nature of rickettsial growth places severe restrictions on the genetic analysis of this distinctive human pathogen. In order to expand the repertoire of genetic tools available for the study of this pathogen, we have employed the versatile mariner-based, Himar1 transposon system to generate insertional mutants of R. prowazekii. A transposon containing the R. prowazekii arr-2 rifampin resistance gene and a gene coding for a green fluorescent protein (GFP(UV)) was constructed and placed on a plasmid expressing the Himar1 transposase. Electroporation of this plasmid into R. prowazekii resulted in numerous transpositions into the rickettsial genome. Transposon insertion sites were identified by rescue cloning, followed by DNA sequencing. Random transpositions integrating at TA sites in both gene coding and intergenic regions were identified. Individual rickettsial clones were isolated by the limiting-dilution technique. Using both fixed and live-cell techniques, R. prowazekii transformants expressing GFP(UV) were easily visible by fluorescence microscopy. Thus, a mariner-based system provides an additional mechanism for generating rickettsial mutants that can be screened using GFP(UV) fluorescence.     

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.     

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.     

Isolation and characterization of the Rickettsia prowazekii recA gene.

The recA gene has been isolated from Rickettsia prowazekii, an obligate intracellular bacterium. Comparison of the amino acid sequence of R. prowazekii RecA with that of Escherichia coli RecA revealed that 62% of the residues were identical. The highest identity was found with RecA of Legionella pneumophila, in which 69% of the residues were identical. Amino acid residues of E. coli RecA associated with functional activities are conserved in rickettsial RecA, and the R. prowazekii recA gene complements E. coli recA mutants for UV light and methyl methanesulfonate sensitivities as well as recombinational deficiencies. The characterized region upstream of rickettsial recA did not contain a sequence homologous to an E. coli LexA binding site (SOS box), suggesting differences in the regulation of the R. prowazekii recA gene.     

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.     

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).     

Differential proteomic analysis of Rickettsia prowazekii propagated in diverse host backgrounds

The obligate intracellular growth of Rickettsia prowazekii places severe restrictions on the analysis of rickettsial gene expression. With a small genome, predicted to code for 835 proteins, identifying which proteins are differentially expressed in rickettsiae that are isolated from different hosts or that vary in virulence is critical to an understanding of rickettsial pathogenicity. We employed a liquid chromatography (LC)-linear trap quadrupole (LTQ)-Orbitrap mass spectrometer for simultaneous acquisition of quantitative mass spectrometry (MS)-only data and tandem mass spectrometry (MS-MS) sequence data. With the use of a combination of commercially available algorithms and in-house software, quantitative MS-only data and comprehensive peptide coverage generated from MS-MS were integrated, resulting in the assignment of peptide identities with intensity values, allowing for the differential comparison of complex protein samples. With the use of these protocols, it was possible to directly compare protein abundance and analyze changes in the total proteome profile of R. prowazekii grown in different host backgrounds. Total protein extracted from rickettsiae grown in murine, tick, and insect cell lines or hen egg yolk sacs was analyzed. Here, we report the fold changes, including an upregulation of shock-related proteins, in rickettsiae cultivated in tissue culture compared to the level for rickettsiae harvested from hen yolk sacs. The ability to directly compare, in a complex sample, differential rickettsial protein expression provides a snapshot of host-specific proteomic profiles that will help to identify proteins important in intracellular growth and virulence.     

Copy number of the 16S rRNA gene in Rickettsia prowazekii.

The obligate intracellular parasite, Rickettsia prowazekii, is a slowly growing bacterium with a doubling time of 8 to 12 h. The copy number of the 16S rRNA gene in the rickettsial chromosome was determined to be one. Genomic DNA from R. prowazekii was digested either by a variety of restriction enzymes known not to cut at any site in the rickettsial 16S rRNA gene or by a combination of these noncutting enzymes and SmaI, which cuts the gene only once. Only one DNA fragment in these digests hybridized to a biotinylated probe containing a portion of the rickettsial 16S rRNA gene. Moreover, the density of the rickettsial 16S rRNA gene fragment after hybridization was equal to the density of each of the seven 16S rRNA gene fragments in Escherichia coli.     

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.     

Identification and initial topological analysis of the Rickettsia prowazekii ATP/ADP translocase.

The Rickettsia prowazekii ATP/ADP translocase was identified by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and immunoblot analysis using antibodies raised against a synthetic peptide corresponding in sequence to the carboxyl-terminal 17 amino acids of the carrier. Both the translocase of R. prowazekii and that expressed by Escherichia coli transformants containing the rickettsial gene had an apparent molecular mass of 36,500 Da by SDS-PAGE analysis, a mass considerably less than that deduced from the sequence of the gene. The SDS-solubilized translocase aggregated upon heating at 100 degrees C in the presence of disulfide bond-reducing agents. Similar concentrations of disulfide bond-reducing agents inhibited the exchange transport of adenine nucleotides by both R. prowazekii and translocase-expressing E. coli. These data suggested that an intramolecular disulfide bond in the translocase was essential for transport activity. The antipeptide antibodies used for identification of the translocase bound preferentially to inside-out membrane vesicles of translocase-expressing E. coli relative to right-side-out spheroplasts, thus indicating that the carboxyl terminus of the carrier is located on the cytoplasmic side of the bacterial inner membrane. Protease studies were unable to localize the carboxyl terminus because of the resistance of this region of the native translocase to proteolytic cleavage. These data in conjunction with hydrophobicity analysis were used to construct an initial topological model of the translocase within the cell membrane.     

Isolation and characterization of the gene coding for the major sigma factor of Rickettsia prowazekii DNA-dependent RNA polymerase.

The gene coding for the major sigma factor of Rickettsia prowazekii, an obligate intracellular parasitic bacterium, has been isolated utilizing an oligodeoxyribonucleotide as a probe to a conserved region of major sigma factors. Nucleotide sequence analysis revealed an open reading frame of 1905 bp that could encode a protein of 635 amino acids (aa) with a calculated molecular size of 73 kDa (sigma 73). R. prowazekii sigma 73 displayed extensive homology with major sigma factors from a variety of eubacteria. Comparison of the major sigma factors from Escherichia coli and R. prowazekii revealed 44.9% aa identity. R. prowazekii sigma 73 produced in E. coli minicells migrated as a 85-kDa protein when analyzed by sodium dodecyl sulfate-polyacrylamide-gel electrophoresis. This anomalous migration is characteristic of eubacterial major sigma factors and agrees with the migration noted for the purified rickettsial sigma protein. Despite a similarity to the E. coli sigma 70 encoded by rpoD, R. prowazekii sigma 73 did not complement E. coli rpoD temperature-sensitive mutants.